DNA encoding a human melanin concentrating hormone receptor (MCH1) and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid encoding a human MCH1 receptor, a purified human MCH1 receptor, vectors comprising isolated nucleic acid encoding a human MCH1 receptor, cells comprising such vectors, antibodies directed to a human MCH1 receptor, nucleic acid probes useful for detecting nucleic acid encoding human MCH1 receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding human MCH1 receptors, transgenic, nonhuman animals which express DNA encoding a normal or mutant human MCH1 receptor, methods of isolating a human MCH1 receptor, methods of treating an abnormality that is linked to the activity of a human MCH1 receptor, as well as methods of determining binding of compounds to mammalian MCH1 receptors.

[0001] This application is a continuation-in-part of U.S. Ser. No.09/224,426, filed Dec. 31, 1998, the contents of which is herebyincorporated by reference into the subject application.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referencedin parentheses by author and year. Full citations for these referencesmay be found at the end of the specification immediately preceding thesequence listings and the claims. The disclosure of these publicationsin their entireties are hereby incorporated by reference into thisapplication to describe more fully the state of the art to which thisinvention pertains.

[0003] Neuroregulators comprise a diverse group of natural products thatsubserve or modulate communication in the nervous system. They include,but are not limited to, neuropeptides, amino acids, biogenic amines,lipids and lipid metabolites, and other metabolic byproducts. Many ofthese neuroregulator substances interact with specific cell surfacereceptors which transduce signals from the outside to the inside of thecell. G-protein coupled receptors (GPCRs) represent a major class ofcell surface receptors with which many neurotransmitters interact tomediate their effects. GPCRs are predicted to have sevenmembrane-spanning domains and are coupled to their effectors viaG-proteins linking receptor activation with intracellular biochemicalsequelae such as stimulation of adenylyl cyclase.

[0004] Melanin-concentrating hormone (MCH) is a cyclic peptideoriginally isolated from salmonid (teleost fish) pituitaries (Kawauchiet al., 1983). In fish the 17 amino acid peptide causes aggregation ofmelanin within the melanophores and inhibits the release of ACTH, actingas a functional antagonist of α-MSH. Mammalian MCH (19 amino acids) ishighly conserved between rat, mouse, and human, exhibiting 100% aminoacid identity, but its physiological roles are less clear. MCH has beenreported to participate in a variety of processes including feeding,water balance, energy metabolism, general arousal/attention state,memory and cognitive functions, and psychiatric disorders (for reviews,see Baker, 1991; Baker, 1994; Nahon, 1994; Knigge et al., 1996). Itsrole in feeding or body weight regulation is supported by a recentNature publication (Qu et al., 1996) demonstrating that MCH isoverexpressed in the hypothalamus of ob/ob mice compared with ob/+ mice,and that fasting further increased MCH mRNA in both obese and normalmice during fasting. MCH also stimulated feeding in normal rats wheninjected into the lateral ventricles (Rossi et al., 1997). MCH also hasbeen reported to functionally antagonize the behavioral effects of α-MSH(Miller et al., 1993; Gonzalez et al, 1996; Sanchez et al., 1997); inaddition, stress has been shown to increase POMC mRNA levels whiledecreasing the MCH precursor preproMCH (ppMCH) mRNA levels (Presse etal., 1992). Thus MCH may serve as an integrative neuropeptide involvedin the reaction to stress, as well as in the regulation of feeding andsexual activity (Baker, 1991; Knigge et al., 1996).

[0005] The gene encoding the MCH precursor (ppMCH) has been cloned andencodes two additional peptides, neuropeptide EI (13 AA) andneuropeptide GE (19AA) (Nahon et al., 1989), which may also havebiological activity. MCH peptide is synthesized primarily inhypothalamic neurons (the zona incerta and lateral hypothalamus) whichproject diffusely to many brain areas and to the pituitary (Bittencourtet al., 1992); NEI has also been identified in medium from explantedhypothalamic neurons (Parkes and Vale, 1993). Localization studies ofthe mRNA indicate that MCH is also present in the periphery (testes andGI tract; Hervieu and Nahon, 1995) but the highest concentrations are inthe hypothalamus. There is also evidence for differentialtissue-dependent processing of proMCH in mammals. A shorter MCH genetranscript that may result from alternate splicing was found in severalbrain areas and peripheral tissues, and a different peptide form wasalso found in the periphery (Viale et al., 1997). In humans, the geneencoding authentic MCH has been localized to chromosome 12, but twocopies of a variant (truncated) gene are present on chromosome 5 (Bretonet al., 1993); the functional significance, if any, of the variant isnot yet known. Finally, the rat MCH gene may encode an additionalputative peptide in a different reading frame (Toumaniantz et al.,1996).

[0006] Although the biological effects of MCH are believed to bemediated by specific receptors, binding sites for MCH have not been welldescribed. A tritiated ligand ([³H]-MCH) was reported to exhibitspecific binding to brain membranes but was unusable for saturationanalyses, so neither affinity nor B_(max) were determined (Drozdz andEberle, 1995). Radioiodination of the tyrosine at position thirteenresulted in a ligand with dramatically reduced biological activity (seeDrozdz and Eberle, 1995). In contrast, the radioiodination of the MCHanalogue (Phe¹³,Tyr¹⁹]-MCH was successful (Drozdz et al., 1995); theligand retained biological activity and exhibited specific binding to avariety of cell lines including mouse melanoma (B16-F1, G4F, and G4F-7),PC12, and COS cells. In G4F-7 cells, the K_(p)=0.118 nM and the B_(max)˜1100 sites/cell. Importantly, the binding was not inhibited by α-MSHbut was weakly inhibited by rat ANF (Ki=116 nM vs. 12 nM for native MCH)(Drozdz et al., 1995). More recently specific MCH binding was reportedin transformed keratinocytes (Burgaud et al., 1997) and melanoma cells(Drozdz et al., 1998), where photo-crosslinking studies suggest that thereceptor is a membrane protein with an apparent molecular weight of45-50 kDaltons, compatible with the molecular weight range of the GPCRsuperfamily of receptors. No radioautoradiographic studies of MCHreceptor localization using this ligand have been reported as yet.

[0007] Signal transduction mechanisms for MCH receptors remain obscure.No direct evidence supporting G-protein coupling exists in mammals, buttwo lines of weak evidence exist in teleost fish for G_(oq)- and/orG_(oi)-type coupling: 1) indirect evidence exists for MCH acting viaphospholipase C in teleost fish melanophores (phospholipase C inhibitorsand protein kinase C inhibitors shift the MCH dose-response curve to theright, and TPA mimics MCH at low doses (Abrao et al., 1991)); and 2)MCH-elicited pigment aggregation in fish melanophores is associated witha reduction in basal cAMP levels, similar to that observed withnorepinephrine (Svensson et al., 1991; Morishita et al., 1993). Arguingagainst G-protein coupling is the general structural homology of MCHwith ANF, whose receptors are not in the GPCR superfamily. Recently theactions of MCH were reported to be mediated via activation of aphosphatidylinositol-3-kinase pathway which is typical of tyrosinekinase and cytokine receptors (Qu et al., 1998); however, since multiplesignaling pathways (receptor cross talk) may produce this mediator noconclusions can be reached regarding MCH signal transduction pathways inmammalian systems.

[0008] The localization and biological activities of MCH peptide suggestthat the modulation of MCH receptor activity may be useful in a numberof therapeutic applications. The role of MCH in feeding is the bestcharacterized of its potential clinical uses. MCH is expressed in thelateral hypothalamus, a brain area implicated in the regulation ofthirst and hunger (Grillon et al., 1997); recently orexins A and B,which are potent orexigenic agents, have been shown to have very similarlocalization to MCH in the lateral hypothalamus (Sakurai et al., 1998).MCH mRNA levels in this brain region are increased in rats after 24hours of food-deprivation (Herve and Feliman, ;997); after insulininjection, a significant increase in the abundance and stainingintensity of MCH immunoreactive perikarya and fibres was observedconcurrent with a significant increase in the level of MCH mRNA(Bahjaoui-Bouhaddi et al., 1994). Consistent with the ability of MCH tostimulate feeding in rats (Rossi et al., 1997) is the observation thatMCH mRNA levels are upregulated in the hypothalami of obese ob/ob mice(Qu et al., 1996), and decreased in the hypothalami of rats treated withleptin, whose food intake and body weight gains are also decreased(Sahu, 1998). MCH appears to act as a functional antagonist of themelanocortin system in its effects on food intake and on hormonesecretion within the HPA (hypothalamopituitary/adrenal axis) (Ludwig etal., 1998). Together these data suggest a role for endogenous MCH in theregulation of energy balance and response to stress, and provide arationale for the development of specific compounds acting at MCHreceptors for use in the treatment of obesity and stress-relateddisorders.

[0009] In all species studied to date, a major portion of the neurons ofthe MCH cell group occupies a rather constant location in those areas ofthe lateral hypothalamus and subthalamus where they lie and may be apart of some of the so-called “extrapyramidal” motor circuits. Theseinvolve substantial striato- and pallidofugal pathways involving thethalamus and cerebral cortex, hypothalamic areas, and reciprocalconnections to subthalamic nucleus, substantia nigra, and mid-braincenters (Bittencourt et al., 1992). In their location, the MCH cellgroup may offer a bridge or mechanism for expressing hypothalamicvisceral activity with appropriate and coordinated motor activity.Clinically it may be of some value to consider the involvement of thisMCH system in movement disorders, such as Parkinson's disease andHuntington's Chorea in which extrapyramidal circuits are known to beinvolved.

[0010] Human genetic linkage studies have located authentic hMCH loci onchromosome 12 (12q23-24) and the variant hMCH loci on chromosome 5(5q12-13) (Pedeutour et al., 1994). Locus 12q23-24 coincides with alocus to which autosomal dominant cerebellar ataxia type II (SCA2) hasbeen mapped (Auburger et al., 1992; Twells et al., 1992). This diseasecomprises neurodegenerative disorders, including an olivopontocerebellaratrophy. Furthermore, the gene for Darier's disease, has been mapped tolocus 12q23-24 (Craddock et al., 1993). Dariers' disease ischaracterized by abnormalities I keratinocyte adhesion and mentalillnesses in some families. In view of the functional andneuroanatomical patterns of the MCH neural system in the rat and humanbrains, the MCH gene may represent a good candidate for SCA2 or Darier'sdisease. Interestingly, diseases with high social impact have beenmapped to this locus. Indeed, the gene responsible for chronic or acuteforms of spinal muscular atrophies has been assigned to chromosome5q12-13 using genetic linkage analysis (Melki et al., 1990; Westbrook etal., 1992). Furthermore, independent lines of evidence support theassignment of a major schizophrenia locus to chromosome 5q11.2-13.3(Sherrington et al., 1988; Bassett et al., 1988; Gilliam et al., 1989).The above studies suggest that MCH may play a role in neurodegenerativediseases and disorders of emotion.

[0011] Additional therapeutic applications for MCH-related compounds aresuggested by the observed effects of MCH in other biological systems.For example, MCH may regulate reproductive functions in male and femalerats. MCH transcripts and MCH peptide were found within germ cells intestes of adult rats, suggesting that MCH may participate in stem cellrenewal and/or differentiation of early spermatocytes (Hervieu et al.,1996). MCH injected directly into the medial preoptic area (MPOA) orventromedial nucleus (VMN) stimulated sexual activity in female rats(Gonzalez et al., 1996). In ovariectomized rats primed with estradiol,MCH stimulated luteinizing hormone (LH) release while anti-MCH antiseruminhibited LH release (Gonzalez et al., 1997). The zona incerta, whichcontains a large population of MCH cell bodies, has previously beenidentified as a regulatory site for the pre-ovulatory LH surge(MacKenzie et al., 1984). MCH has been reported to influence release ofpituitary hormones including ACTH and oxytocin. MCH analogues may alsobe useful in treating epilepsy. In the PTZ seizure model, injection ofMCH prior to seizure induction prevented seizure activity in both ratsand guinea pigs, suggesting that MCH-containing neurons may participatein the neural circuitry underlying PTZ-induced seizure (Knigge andWagner, 1997). MCH has also been observed to affect behavioralcorrelates of cognitive functions. MCH treatment hastened extinction ofthe passive avoidance response in rats (McBride et al., 1994), raisingthe possibility that MCH receptor antagonists may be beneficial formemory storage and/or retention. A possible role for MCH in themodulation or perception of pain is supported by the dense innervationof the periaqueductal grey (PAG) by MCH-positive fibers. Finally, MCHmay participate in the regulation of fluid intake. ICV infusion of MCHin conscious sheep produced diuretic, natriuretic, and kaliureticchanges in response to increased plasma volume (Parkes, 1996). Togetherwith anatomical data reporting the presence of MCH in fluid regulatoryareas of the brain, the results indicate that MCH may be an importantpeptide involved in the central control of fluid homeostasis in mammals.

SUMMARY OF THE INVENTION

[0012] This invention provides an isolated nucleic acid encoding a humanMCH1 receptor or a mutant of such human MCH1 receptor which is activatedby MCH or an analog or homolog thereof.

[0013] This invention provides a nucleic acid encoding a human MCH1receptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in FIG. 1 (SEQ ID NO: 1) under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when an MCH1 ligand is added to the culture and theCHO cells contain the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement.

[0014] This invention provides a purified human MCH1 receptor protein.

[0015] This invention provides a vector comprising a nucleic acidencoding a human MCH1 receptor, particularly a vector adapted forexpression of the human MCH1 receptor in mammalian or non-mammaliancells. One such vector is a plasmid designated pEXJ.HR-TL231 (ATCCAccession No. 203197) which comprises a nucleotide sequence encoding ahuman MCH1 receptor.

[0016] This invention also provides a cell comprising a vector whichcomprises a nucleic acid encoding a human MCH1 receptor as well as amembrane preparation isolated from such cells.

[0017] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides which specifically hybridizes with a nucleicacid encoding a mammalian MCH1 receptor, wherein the probe has a uniquesequence corresponding to a sequence present within the nucleic acidwhich encodes the human MCH1 receptor or its complement, both of whichare present n plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197).

[0018] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides which specifically hybridizes with a nucleicacid encoding a mammalian MCH1 receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in FIG. 1 (SEQ ID NO: 1) or (b) the reverse complementthereof.

[0019] This invention also provides an antisense oligonucleotide havinga sequence capable of specifically hybridizing an RNA encoding a humanMCH1 receptor, so as to prevent translation of the RNA and an antisenseoligonucleotide having a sequence capable of specifically hybridizing tothe genomic DNA encoding a human MCH1 receptor.

[0020] This invention further provides an antibody capable of binding toa human MCH1 receptor as well as an agent capable of competitivelyinhibiting the binding of the antibody to a human MCH1 receptor.

[0021] This invention provides a pharmaceutical composition comprising(a) an amount of the oligonucleotide described above capable of passingthrough a cell membrane and effective to reduce expression of a humanMCH1 receptor and (b) a pharmaceutically acceptable carrier capable ofpassing through the cell membrane.

[0022] Moreover, this invention provides a transgenic, nonhuman mammalexpressing DNA encoding a human MCH1 receptor. This invention alsoprovides a transgenic, nonhuman mammal comprising a homologousrecombination knockout of the native human MCH1 receptor. This inventionfurther provides a transgenic, nonhuman mammal whose genome comprisesantisense DNA complementary to the DNA encoding a human MCH1 receptor soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the human MCH1 receptor and whichhybridizes to mRNA encoding the human MCH1 receptor, thereby reducingits translation.

[0023] In one embodiment this invention provides a process foridentifying a chemical compound which specifically binds to a mammalianMCH1 receptor which comprises contacting cells containing DNA encodingand expressing on their cell surface a mammalian MCH1 receptor, whereinsuch cells do not normally express the mammalian MCH1 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the mammalian MCH1 receptor.

[0024] This invention provides a process for identifying a chemicalcompound which specifically binds to a mammalian MCH1 receptor whichcomprises contacting a membrane preparation from cells transfected withDNA encoding and expressing on their cell surface the mammalian MCH1receptor, wherein such cells do not normally express the mammalian MCH1receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianMCH1 receptor.

[0025] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian MCH1 receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian MCH1 receptor, whereinsuch cells do not normally express the mammalian MCH1 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the mammalian MCH1receptor, a decrease in the binding of the second chemical compound tothe mammalian MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian MCH1receptor.

[0026] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian MCH1 receptor which comprises separately contacting a membranefraction from a cell extract of cells expressing on their cell surfacethe mammalian MCH1 receptor, wherein such cells do not normally expressthe mammalian MCH1 receptor, with both the chemical compound and asecond chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the mammalian MCH1 receptor, a decrease in the binding of the secondchemical compound to the mammalian MCH1 receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian MCH1 receptor.

[0027] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian MCH1 receptor toidentify a compound which specifically binds to the mammalian MCH1receptor, which comprises (a) contacting cells transfected with andexpressing DNA encoding the mammalian MCH1 receptor with a compoundknown to bind specifically to the mammalian MCH1 receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the mammalian MCH1 receptor, underconditions permitting binding of compounds known to bind the mammalianMCH1 receptor; (c) determining whether the binding of the compound knownto bind to the mammalian MCH1 receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian MCH1 receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian MCH1receptor.

[0028] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian MCH1 receptor toidentify a compound which specifically binds to the mammalian MCH1receptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing DNA encoding a mammalian MCH1receptor with a compound known to bind specifically to the mammalianMCH1 receptor; (b) contacting the preparation of step (a) with theplurality of compounds not known to bind specifically to the mammalianMCH1 receptor, under conditions permitting binding of compounds known tobind the mammalian MCH1 receptor; (c) determining whether the binding ofthe compound known to bind to the mammalian MCH1 receptor is reduced inthe presence of the compounds within the plurality of compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to themammalian MCH1 receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian MCH1 receptor.

[0029] This invention provides a method of detecting expression of amammalian MCH1 receptor by detecting the presence of mRNA coding for themammalian MCH1 receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained with a nucleic acid probe underhybridizing conditions, detecting the presence of mRNA hybridizing tothe probe, and thereby detecting the expression of the mammalian MCH1receptor by the cell.

[0030] This invention provides a method of detecting the presence of amammalian MCH1 receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianMCH1 receptor on the surface of the cell.

[0031] This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a transgenic, nonhuman mammal whose levels of humanMCH1 receptor activity are varied by use of an inducible promoter whichregulates human MCH1 receptor expression.

[0032] This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a panel of transgenic, nonhuman mammals eachexpressing a different amount of human MCH1 receptor.

[0033] This invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a human MCH1 receptorcomprising administering a compound to the transgenic, nonhuman mammaland determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal asa result of overactivity of a human MCH1 receptor, the alleviation ofthe abnormality identifying the compound as an antagonist. Thisinvention also provides an antagonist identified by this method. Thisinvention further provides a pharmaceutical composition comprising anantagonist identified by this method and a pharmaceutically acceptablecarrier.

[0034] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a human MCH1 receptor which comprises administering to the subject aneffective amount of this pharmaceutical composition, thereby treatingthe abnormality.

[0035] This invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a human MCH1receptor comprising administering a compound to a transgenic, nonhumanmammal, and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by thismethod. This invention further provides a pharmaceutical compositioncomprising an agonist identified by this method and a pharmaceuticallyacceptable carrier. This invention provides a method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a human MCH1 receptor which comprisesadministering to the subject an effective amount of this pharmaceuticalcomposition, thereby treating the abnormality.

[0036] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific mammalianallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human MCH1 receptor and labeled with a detectable marker; (e)detecting labeled bands which have hybridized to the DNA encoding ahuman MCH1 receptor labeled with a detectable marker to create a uniqueband pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same.

[0037] This invention provides a method of preparing a purified humanMCH1 receptor which comprises: (a) inducing cells to express the humanMCH1 receptor; (b) recovering the human MCH1 receptor from the inducedcells; and (c) purifying the human MCH1 receptor so recovered.

[0038] This invention provides a method of preparing a purified humanMCH1 receptor which comprises: (a)inserting nucleic acid encoding thehuman MCH1 receptor in a suitable vector; (b) introducing the resultingvector in a suitable host cell; (c) placing the resulting cell insuitable condition permitting the production of the isolated human MCH1receptor; (d) recovering the human MCH1 receptor produced by theresulting cell; and (e) purifying the human MCH1 receptor so recovered.

[0039] This invention provides a process for determining whether achemical compound is a mammalian MCH1 receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian MCH1 receptor with the compound under conditions permittingthe activation of the mammalian MCH1 receptor, and detecting an increasein mammalian MCH1 receptor activity, so as to thereby determine whetherthe compound is a mammalian MCH1 receptor agonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian MCH1 receptor agonist determined by this process effective toincrease activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

[0040] This invention provides a process for determining whether achemical compound is a mammalian MCH1 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian MCH1 receptor with the compound in the presence of a knownmammalian MCH1 receptor agonist, under conditions permitting theactivation of the mammalian MCH1 receptor, and detecting a decrease inmammalian MCH1 receptor activity, so as to thereby determine whether thecompound is a mammalian MCH1 receptor antagonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian MCH1 receptor antagonist determined by this process effectiveto reduce activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

[0041] This invention provides a process for determining whether achemical compound specifically binds to and activates a mammalian MCH1receptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the mammalian MCH1receptor, wherein such cells do not normally express the mammalian MCH1receptor, with the chemical compound under conditions suitable foractivation of the mammalian MCH1 receptor, and measuring the secondmessenger response in the presence and in the absence of the chemicalcompound, a change in the second messenger response in the presence ofthe chemical compound indicating that the compound activates themammalian MCH1 receptor. This invention also provides a compounddetermined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (aMCH1 receptor agonist) determined by this process effective to increaseactivity of a mammalian MCH1 receptor and a pharmaceutically acceptablecarrier.

[0042] This invention provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian MCH1 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian MCH1 receptor, wherein such cells do not normallyexpress the mammalian MCH1 receptor, with both the chemical compound anda second chemical compound known to activate the mammalian MCH1receptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian MCH1 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian MCH1 receptor. This invention also providesa compound determined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian MCH1 receptor antagonist) determined by this effective toreduce activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

[0043] This invention provides a method of screening a plurality ofchemical compounds not known to activate a mammalian MCH1 receptor toidentify a compound which activates the mammalian MCH1 receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian MCH1 receptor with the plurality of compounds not known toactivate the mammalian MCH1 receptor, under conditions permittingactivation of the mammalian MCH1 receptor; (b) determining whether theactivity of the mammalian MCH1 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the mammalian MCH1 receptor is increased by each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which activates the mammalian MCH1 receptor. This inventionalso provides a compound identified by this method. This inventionfurther provides a pharmaceutical composition which comprises an amountof the compound (a mammalian MCH1 receptor agonist) identified by thismethod effective to increase activity of a mammalian MCH1 receptor and apharmaceutically acceptable carrier.

[0044] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a mammalianMCH1 receptor to identify a compound which inhibits the activation tothe mammalian MCH1 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian MCH1 receptor with theplurality of compounds in the presence of a known mammalian MCH1receptor agonist, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activation of themammalian MCH1 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian MCH1 receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian MCH1 receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian MCH1 receptor. This invention also provides a compoundidentified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian MCH1 receptor antagonist) identified by this process effectiveto decrease activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

[0045] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a mammalian MCH1 receptor which comprises administering to thesubject an amount of a compound which is a mammalian MCH1 receptoragonist effective to treat the abnormality.

[0046] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a mammalian MCH1 receptor which comprises administering to thesubject an amount of a compound which is a mammalian MCH1 receptorantagonist effective to treat the abnormality.

[0047] This invention provides a process for making a composition ofmatter which specifically binds to a mammalian MCH1 receptor whichcomprises identifying a chemical compound using any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian MCH1 receptor and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof this invention further provides a process forpreparing a pharmaceutical composition which comprises administering apharmaceutically acceptable carrier and a pharmaceutically acceptableamount of a chemical compound identified by any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian MCH1 receptor or a novelstructural and functional analog or homolog thereof.

[0048] This invention provides a process for determining whether achemical compound is a human MCH1 receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding the humanMCH1 receptor with the compound in the presence of a known human MCH1receptor agonist, under conditions permitting the activation of thehuman MCH1 receptor, and detecting a decrease in human MCH1 receptoractivity, so as to thereby determine whether the compound is a humanMCH1 receptor antagonist, wherein the DNA encoding the human MCH1receptor comprises the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH,and the cells do not express the MCH1 receptor prior to transfectingthem.

[0049] This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of ahuman MCH1 receptor, which comprises separately contacting cellsexpressing on their cell surface the human MCH1 receptor and producing asecond messenger response upon activation of the human MCH1 receptor,wherein such cells do not normally express the human MCH1 receptor andthe DNA encoding the human MCH1 receptor comprises the sequence shown inFIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197), with both the chemical compound and a secondchemical compound known to activate the human MCH1 receptor, and withonly the second chemical compound, under conditions suitable foractivation of the human MCH1 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of the humanMCH1 receptor, wherein the second chemical compound is MCH or a homologor analog of MCH.

[0050] This invention further provides a method of screening a pluralityof chemical compounds not known to inhibit the activation of a humanMCH1 receptor to identify a compound which inhibits the activation ofthe human MCH1 receptor, which comprises:

[0051] (a) contacting cells transfected with and expressing the humanMCH1 receptor, wherein such cells do not normally express the human MCH1receptor and the DNA encoding the human MCH1 receptor comprises thesequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), with the plurality ofcompounds in the presence of a known human MCH1 receptor agonist, underconditions permitting activation of the human MCH1 receptor, wherein theknown MCH1 receptor agonist is MCH or a homolog or analog of MCH;

[0052] (b) determining whether the activation of the human MCH1 receptoris reduced in the presence of the plurality of compounds, relative tothe activation of the human MCH1 receptor in the absence of theplurality of compounds; and if so

[0053] (c) separately determining the extent of inhibition of activationof the human MCH1 receptor for each compound included in the pluralityof compounds, so as to thereby identify the compound which inhibits theactivation of the human MCH1 receptor.

[0054] This invention provides a process for making a composition ofmatter which specifically binds to a human MCH1 receptor which comprisesidentifying a chemical compound which specifically binds to the humanMCH1 receptor and then synthesizing the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting cellsexpressing on their cell surface the human MCH1 receptor, with both thechemical compound and a second chemical compound known to bind to thereceptor, and separately with only the second chemical compound, underconditions suitable for binding of both compounds, and detecting theextent of specific binding of the chemical compound to the human MCH1receptor, a decrease in the binding of the second chemical compound tothe human MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the human MCH1 receptor,wherein the cells do not normally express the human MCH1 receptor, thehuman MCH1 receptor is encoded by nucleic acid comprising the sequenceshown in FIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231(ATCC Accession No. 203197), and the second chemical compound is MCH ora homolog or analog of MCH.

[0055] This invention further provides a process for making acomposition of matter which specifically binds to a human MCH1 receptorwhich comprises identifying a chemical compound which specifically bindsto the human MCH1 receptor and then synthesizing the chemical compoundor a structural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting amembrane preparation from cells expressing on their cell surface thehuman MCH1 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and separately withonly the second chemical compound, under conditions suitable for bindingof both compounds, and detecting the extent of specific binding of thechemical compound to the human MCH1 receptor, a decrease in the bindingof the second chemical compound to the human MCH1 receptor in thepresence of the chemical compound indicating that the chemical compoundbinds to the human MCH1 receptor, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and thesecond chemical compound is MCH or a homolog or analog of MCH.

[0056] This invention also provides a process for making a compositionof matter which is a human MCH1 receptor antagonist which comprisesidentifying a chemical compound which is a human MCH1 receptorantagonist and then synthesizing the chemical compound or a structuraland functional analog or homolog thereof, wherein the chemical compoundis identified as a human MCH1 receptor antagonist by a process whichcomprises contacting cells transfected with and expressing DNA encodingthe human MCH1 receptor with the compound in the presence of a knownhuman MCH1 receptor agonist, under conditions permitting the activationof the human MCH1 receptor, and detecting a decrease in human MCH1receptor activity, so as to thereby determine whether the compound is ahuman MCH1 receptor antagonist, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH.

[0057] This inventions still further provides a process for making acomposition of matter which specifically binds to and inhibits theactivation of a human MCH1 receptor which comprises identifying achemical compound which specifically binds to and inhibits theactivation of the human MCH1 receptor and then synthesizing the chemicalcompound or a structural and functional analog or homolog thereof,wherein the chemical compound is identified as binding to and inhibitingthe activation of the human MCH1 receptor by a process which comprisesseparately contacting cells expressing on their cell surface the humanMCH1 receptor and producing a second messenger response upon activationof the human MCH1 receptor, wherein such cells do not normally expressthe human MCH1 receptor and the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), withboth the chemical compound and a second chemical compound known toactivate the human MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the human MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the human MCH1 receptor, wherein the second chemicalcompound is MCH or a homolog or analog of MCH.

[0058] This invention provides a process for preparing a compositionwhich comprises identifying a chemical compound which specifically bindsto a human MCH1 receptor, and then admixing a carrier and the chemicalcompound or a structural and functional analog or homolog thereof,wherein the chemical compound is identified as binding to the human MCH1receptor by a process involving competitive binding which comprisescontacting cells expressing on their cell surface the human MCH1receptor, with both the chemical compound and a second chemical compoundknown to bind to the receptor, and separately with only the secondchemical compound, under conditions suitable for binding of bothcompounds, and detecting the extent of specific binding of the chemicalcompound to the human MCH1 receptor, a decrease in the binding of thesecond chemical compound to the human MCH1 receptor in the presence ofthe chemical compound indicating that the chemical compound binds to thehuman MCH1 receptor, wherein the cells do not normally express the humanMCH1 receptor, the human MCH1 receptor is encoded by nucleic acidcomprising the sequence shown in FIG. 1 (Seq. ID No. 1) or contained inplasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and the secondchemical compound is MCH or a homolog or analog of MCH.

[0059] This invention further provides a process for preparing acomposition which comprises identifying a chemical compound whichspecifically binds to a human MCH1 receptor, and then admixing a carrierand the chemical compound or a structural and functional analog orhomolog thereof, wherein the chemical compound is identified as bindingto the human MCH1 receptor by a process involving competitive bindingwhich comprises contacting a membrane preparation from cells expressingon their cell surface the human MCH1 receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and separately with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting the extent ofspecific binding of the chemical compound to the human MCH1 receptor, adecrease in the binding of the second chemical compound to the humanMCH1 receptor in the presence of the chemical compound indicating thatthe chemical compound binds to the human MCH1 receptor, wherein thecells do not normally express the human MCH1 receptor, the human MCH1receptor is encoded by nucleic acid comprising the sequence shown inFIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197), and the second chemical compound is MCH or ahomolog or analog of MCH.

[0060] This invention also provides a process for preparing acomposition which comprises identifying a chemical compound which is ahuman MCH1 receptor antagonist, and then admixing a carrier and thechemical compound or a structural and functional analog or homologthereof, wherein the chemical compound is identified as a human MCH1receptor antagonist by a process which comprises contacting cellstransfected with and expressing DNA encoding the human MCH1 receptorwith the compound in the presence of a known human MCH1 receptoragonist, under conditions permitting the activation of the human MCH1receptor, and detecting a decrease in human MCH1 receptor activity, soas to thereby determine whether the compound is a human MCH1 receptorantagonist, wherein the cells do not normally express the human MCH1receptor, the human MCH1 receptor is encoded by nucleic acid comprisingthe sequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), and the known human MCH1receptor agonist is MCH or a homolog or analog of MCH.

[0061] This invention still further provides a process for preparing acomposition which comprises identifying a chemical compound whichspecifically binds to and inhibits the activation of a human MCH1receptor, and then admixing a carrier and the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to and inhibiting activationof the human MCH1 receptor by a process which comprises separatelycontacting cells expressing on their cell surface the human MCH1receptor and producing a second messenger response upon activation ofthe human MCH1 receptor, wherein such cells do not normally express thehuman MCH1 receptor and the human MCH: receptor is encoded by nucleicacid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmic pEXJ.HR-TL231 (ATCC Accession No. 203197), withboth the chemical compound and a second chemical compound known toactivate the human MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the human MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the human MCH1 receptor, wherein the second chemicalcompound is MCH or a homolog or analog of MCH.

BRIEF DESCRIPTION OF THE FIGURES

[0062]FIG. 1

[0063] Nucleotide sequence encoding a human MCH1 receptor (MCH1) (SEQ IDNO: 1). Three potential start (ATG) codons and the stop (TGA) codon areunderlined.

[0064]FIG. 2

[0065] Deduced amino acid sequence (SEQ ID NO: 2) of the human MCH1receptor (MCH1) encoded by the nucleotide sequence shown FIG. 1 (SEQ IDNO: 1).

[0066]FIG. 3

[0067] Deduced amino acid sequence for human MCH1 (SEQ ID NO: 2). Theseven putative transmembrane (TM) regions are underlined.

[0068]FIG. 4

[0069] Nucleotide sequence of rat MCH1 (SEQ ID NO: 3). One start (ATG)codon and the stop codon (TGA) are underlined.

[0070]FIG. 5

[0071] Deduced amino acid sequence for rat MCH1 (SEQ ID NO: 4).

[0072]FIG. 6

[0073] MCH1-mediated PI dose response to MCH.

[0074]FIG. 7

[0075] MCH1 challenge with several compounds of interest.

[0076]FIG. 8

[0077] MCH1-mediated extracellular acidification response to MCH andPhe¹³,Tyr¹⁹-MCH. Results are reported as the average of two independentexperiments performed in duplicate.

[0078]FIG. 9

[0079] Transcriptional response of MCH1-transfected Cos-7 cells to MCH.

[0080]FIG. 10

[0081] Binding of [¹²⁵I]Phe¹³,Tyr¹⁹-MCH on MCH1-transfected Cos-7 cellmembranes. Results are means ±S.E.M. (vertical lines) of triplicatedeterminations.

[0082]FIG. 11

[0083] RT-PCR detection of MCH1 receptor mRNA in human mRNA samples.

[0084]FIG. 12

[0085] Amino acid alignment of the N-terminal regions of MCH1 receptorsencoded by various plasmids. The mutations present in R106 (SEQ ID NO:16) and R114 (SEQ ID NO: 17) are shown in lower case. Potentialinitiating methionines are shown in bold. The amino acid sequencedownstream of position 100 is identical for all four plasmids.

[0086]FIG. 13

[0087] Amino acid sequence (SEQ ID NO: 27) of the mutant human MCH1receptor encoded by plasmid R106.

[0088]FIG. 14

[0089] Amino acid sequence (SEQ ID NO: 28) of the mutant human MCH1receptor encoded by plasmid R114.

[0090]FIG. 15

[0091] Amino acid sequence (SEQ ID NO: 29) of the mutant human MCH1receptor encoded by plasmid BO120.

DETAILED DESCRIPTION OF THE INVENTION

[0092] Throughout this application, the following standard abbreviationsare used to indicate specific nucleotide bases:

[0093] A=adenine

[0094] G=guanine

[0095] C=cytosine

[0096] T=thymine

[0097] U=uracil

[0098] M=adenine or cytosine

[0099] R=adenine or guanine

[0100] w=adenine, thymine, or uracil

[0101] S=cytosine or guanine

[0102] Y=cytosine, thymine, or uracil

[0103] K=guanine, thymine, or uracil

[0104] V=adenine, cytosine, or guanine (not thymine or uracil

[0105] H=adenine, cytosine, thymine, or uracil (not guanine)

[0106] D=adenine, guanine, thymine, or uracil (not cytosine)

[0107] B=cytosine, guanine, thymine, or uracil (not adenine)

[0108] N=adenine, cytosine, guanine, thymine, or uracil (or othermodified base such as inosine)

[0109] I=inosine

[0110] Furthermore, the term “agonist” is used throughout thisapplication to indicate any peptide or non-peptidyl compound whichincreases the activity of any of the polypeptides of the subjectinvention. The term “antagonist” is used throughout this application toindicate any peptide or non-peptidyl compound which decreases theactivity of any of the polypeptides of the subject invention. The term“mammalian” is used throughout this invention to include mutant forms ofthe human MCH1 receptor.

[0111] The activity of a G-protein coupled receptor such as thepolypeptides disclosed herein may be measured using any of a variety offunctional assays in which activation of the receptor in questionresults in an observable change in the level of some second messengersystem, including, but not limited to, adenylate cyclase, calciummobilization, arachidonic acid release, ion channel activity, inositolphospholipid hydrolysis or guanylyl cyclase. Heterologous expressionsystems utilizing appropriate host cells to express the nucleic acid ofthe subject invention are used to obtain the desired second messengercoupling. Receptor activity may also be assayed in an oocyte expressionsystem.

[0112] It is possible that the human MCH1 receptor gene contains intronsand furthermore, the possibility exists that additional introns couldexist in coding or non-coding regions. In addition, spliced form(s) ofmRNA may encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene. (Burns et al.,1996; Chu et al., 1996). Such variants may exhibit pharmacologicproperties differing from the polypeptide encoded by the original gene.

[0113] This invention provides splice variants of the human MCH1receptor disclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding the human MCH1 receptor of this invention.

[0114] The nucleic acid of the subject invention also includes nucleicacid analogs of the human MCH1 receptor gene, wherein the human MCH1receptor gene comprises the nucleic acid sequence shown in FIG. 1 orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197). Nucleicacid analogs of the human MCH1 receptor genes differ from the human MCH1receptor gene described herein in terms of the identity or location ofone or more nucleic acid bases (deletion analogs containing less thanall of the nucleic acid bases shown in FIG. 1 or contained in plasmidpEXJ.HR-TL231, substitution analogs wherein one or more nucleic acidbases shown in FIG. 1 or contained in plasmids pEXJ.HR-TL231 arereplaced by other nucleic acid bases, and addition analogs, wherein oneor more nucleic acid bases are added to a terminal or medial portion ofthe nucleic acid sequence) and which encode proteins which share some orall of the properties of the proteins encoded by the nucleic acidsequences shown in FIG. 1 or contained in plasmid pEXJ.HR-TL231. In oneembodiment of the present invention, the nucleic acid analog encodes aprotein which comprises an amino acid sequence as shown in FIG. 2 orencoded by the nucleic acid sequence contained in plasmid pEXJ.HR-TL231.In another embodiment, the nucleic acid analog encodes a proteincomprising an amino acid sequence which differs from the amino acidsequences shown in FIG. 2 or encoded by the nucleic acid contained inplasmids pEXJ.HR-TL231. In a further embodiment, the protein encoded bythe nucleic acid analog has a function which is the same as the functionof the receptor protein comprising the amino acid sequence shown in FIG.2. In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteincomprising the amino acid sequence shown in FIG. 2. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

[0115] This invention provides the above-described isolated nucleicacid, wherein the nucleic acid is DNA. In an embodiment, the DNA iscDNA. In another embodiment, the DNA is genomic DNA. In still anotherembodiment, the nucleic acid is RNA. Methods for production andmanipulation of nucleic acid molecules are well known in the art.

[0116] This invention further provides nucleic acid which is degeneratewith respect to the DNA encoding the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotides sequence shown in FIG. 1 (SEQID NO: 2) or the nucleotide sequence contained in the plasmidpEXJ.HR-TL231, that is, a nucleotide sequence which is translated intothe same amino acid sequence.

[0117] This invention also encompasses DNAs and cDNAs which encode aminoacid sequences which differ from those of the polypeptides of thisinvention, but which should not produce phenotypic changes. Alternately,this invention also encompasses DNAs, cDNAs, and RNAs which hybridize tothe DNA, cDNA, and RNA of the subject invention. Hybridization methodsare well known to those of skill in the art.

[0118] The nucleic acids of the subject invention also include nucleicacid molecules coding for polypeptide analogs, fragments or derivativesof antigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors. Thecreation of polypeptide analogs is well known to those of skill in theart (R. F. Spurney et al. (1997); Fong, T. M. et al. (1995); Underwood,D. J. et al. (1994); Graziano, M. P. et al. (1996); Guan X. M. et al.(1995)).

[0119] The modified polypeptides of this invention may be transfectedinto cells either transiently or stably using methods well-known in theart, examples of which are disclosed herein. This invention alsoprovides for binding assays using the modified polypeptides, in whichthe polypeptide is expressed either transiently or in stable cell lines.This invention further provides a compound identified using a modifiedpolypeptide in a binding assay such as the binding assays describedherein.

[0120] The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

[0121] This invention provides an isolated nucleic acid encoding a humanMCH1 receptor or a mutant of such human MCH1 receptor which is activatedby MCH or an analog or homolog thereof. In one embodiment, the nucleicacid is DNA. In another embodiment, the DNA is cDNA. In anotherembodiment, the DNA is genomic DNA. In another embodiment, the nucleicacid is RNA.

[0122] This invention also provides methods of using an isolated nucleicacid encoding species homologs of the MCH1 receptor encoded by thenucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) or encoded by theplasmid pEXJ.HR-TL231. In one embodiment, the nucleic acid encodes amammalian MCH1 receptor homolog which has substantially the same aminoacid sequence as does the MCH1 receptor encoded by the plasmidpEXJ.HR-TL231. In another embodiment, the nucleic acid encodes amammalian MCH1 receptor homolog which has above 65% amino acid identityto the MCH1 receptor encoded by the plasmid pEXJ.HR-TL231; preferablyabove 75% amino acid identity to the MCH1 receptor encoded by theplasmid pEXJ.HR-TL231; more preferably above 85% amino acid identity tothe MCH1 receptor encoded by the plasmid pEXJ.HR-TL231; most preferablyabove 95% amino acid identity to the MCH1 receptor encoded by theplasmid pEXJ.HR-TL231. In another embodiment, the mammalian MCH1receptor homolog has above 70% nucleic acid identity to the MCH1receptor gene contained in plasmid pEXJ.HR-TL231; preferably above 80%nucleic acid identity to the MCH1 receptor gene contained in the plasmidpEXJ.HR-TL231; more preferably above 90% nucleic acid identity to theMCH1 receptor gene contained in the plasmid pEXJ.HR-TL231. Examples ofmethods for isolating and purifying species homologs are describedelsewhere (e.g., U.S. Pat. No. 5,602,024, WO94/14957, WO97/26853,WO98/15570).

[0123] In a separate embodiment of the present invention, the nucleicacid encodes a MCH1 receptor which has an amino acid sequence identicalto that encoded by the plasmid pEXJ.HR-TL231. In a further embodiment,the MCH1 receptor comprises a sequence substantially the same as theamino acid sequence shown in FIG. 2 (SEQ ID NO: 2). In anotherembodiment, the MCH1 receptor comprises an amino acid sequence as shownin FIG. 2 (SEQ ID NO: 2).

[0124] In one embodiment, the mutant human MCH1 receptor comprises anamino acid sequence as shown in FIG. 13 (SEQ ID NO: 27). In anotherembodiment, the mutant human MCH1 receptor comprises an amino acidsequence as shown in FIG. 14 (SEQ ID NO: 28). In still anotherembodiment, the mutant human MCH1 receptor comprises an amino acidsequence as shown in FIG. 15 (SEQ ID NO: 29).

[0125] In separate embodiments, the human MCH1 receptor is encoded bythe nucleic acid sequence shown in FIG. 1 beginning with any of thethree indicated start (ATG) codons.

[0126] This invention provides an isolated nucleic acid encoding amodified human MCH1 receptor, which differs from a human MCH1 receptorby having an amino acid(s) deletion, replacement, or addition in thethird intracellular domain.

[0127] This invention provides a nucleic acid encoding a human MCH1receptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in FIG. 1 (SEQ ID NO: 1) under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a MCH1 ligand is added to the culture and theCHO cells contain the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement. Hybridization at lowstringency is performed at 40° C. in a hybridization buffer containing25% formamide, 5×SCC, 7 mM Tris, 1×Denhardt's, 25 μl/ml salmon spermDNA. Wash at 40° C. in 0.1×SCC, 0.1% SDS. Changes in pH are measuredthrough microphysiometric measurement of receptor mediated extracellularacidification rates. Because cellular metabolism is intricately involvedin a broad range of cellular events (including receptor activation ofmultiple messenger pathways), the use of microphysiometric measurementsof cell metabolism can in principle provide a generic assay of cellularactivity arising from the activation of any receptor regardless of thespecifics of the receptor's signaling pathway. General guidelines fortransient receptor expression, cell preparation and microphysiometricrecording are described elsewhere (Salon, J. A. and Owicki, J. A.,1996). Receptors and/or control vectors are transiently expressed inCHO-K1 cells, by liposome mediated transfection according to themanufacturers recommendations (LipofectAMINE, GibcoERL, Gaithersburg,Md.), and maintained in Ham's F-12 complete (10% serum). A total of 10 gof DNA is used to transfect each 75 cm² flask which had been split 24hours prior to the transfection and judged to be 70-80% confluent at thetime of transfection. 24 hours post transfection, the cells areharvested and 3×10⁵ cells seeded into microphysiometer capsules. Cellsare allowed to attach to the capsule membrane for an additional 24hours; during the last 16 hours, the cells are switched to serum-freeF-12 complete to minimize ill-defined metabolic stimulation caused byassorted serum factors. On the day of the experiment the cell capsulesare transferred to the microphysiometer and allowed to equilibrate inrecording media (low buffer RPMI 1640, no bicarbonate, no serum(Molecular Devices Corporation, Sunnyvale, Calif.) containing 0.1% fattyacid free BSA), during which a baseline measurement of basal metabolicactivity is established. A standard recording protocol specifies a 100μl/min flow rate, with a 2 min total pump cycle which includes a 30 secflow interruption during which the acidification rate measurement istaken. Ligand challenges involve a 1 min 20 sec exposure to the samplejust prior to the first post challenge rate measurement being taken,followed by two additional pump cycles for a total of 5 min 20 secsample exposure. Typically, drugs in a primary screen are presented tothe cells at 10 μM final concentration. Ligand samples are then washedout and the acidification rates reported are expressed as a percentageincrease of the peak response over the baseline rate observed just priorto challenge. An examples of a MCH ligand includes, but is not limitedto, the endogenous MCH peptide.

[0128] This invention provides a purified human MCH1 receptor protein.

[0129] This invention provides a vector comprising nucleic acid encodinga human MCH1 receptor. In an embodiment, the vector is adapted forexpression in a cell which comprises the regulatory elements necessaryfor expression of the nucleic acid in the cell operatively linked to thenucleic acid encoding the human MCH1 receptor as to permit expressionthereof. In separate embodiments, the cell is a bacterial cell, anamphibian cell, a yeast cell, an insect cell or a mammalian cell. Inanother embodiment, the vector is a baculovirus. In one embodiment, thevector is a plasmid.

[0130] This invention provides a plasmid designated pEXJ.HR-TL231 (ATCCAccession No. 203197). This plasmid comprises the regulatory elementsnecessary for expression of DNA in a mammalian cell operatively linkedto -DNA encoding the human MCH1 receptor so as to permit expressionthereof. This plasmid (pEXJ.HR-TL231) was deposited on Sep. 17, 1998,with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.203197.

[0131] This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the polypeptidedepending upon the host cell used. In an embodiment, the vector orplasmid comprises the coding sequence of the polypeptide and theregulatory elements necessary for expression in the host cell.

[0132] This invention provides a cell comprising a vector comprising anucleic acid encoding the human MCH1 receptor. In an embodiment, thecell is a non-mammalian cell. In a further embodiment, the non-mammalia,cell is a Xenopus oocyte cell or a Xenopus melanophore cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, themammalian cell is a COS-7 cell, a 293 human embryonic kidney cell, aNIH-3T3 cell, a LM(tk-) cell, a mouse Y1 cell, or a CHO cell.

[0133] This invention provides an insect cell comprising a vectoradapted for expression in an insect cell which comprises a nucleic acidencoding a human MCH1 receptor. In another embodiment, the insect cellis an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B1-4 (HighFive) cell.

[0134] This invention provides a membrane preparation isolated from anyone of the cells described above.

[0135] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a human MCH1 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding a human MCH1 receptor present in plasmidpEXJ.HR-TL231. This invention also provides a nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a human MCH1 receptor, wherein the probehas a unique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) or (b) the reversecomplement thereto. In one embodiment, the nucleic acid is DNA. Inanother embodiment, the nucleic acid is RNA.

[0136] As used herein, the phrase “specifically hybridizing” means theability of a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

[0137] Nucleic acid probe technology is well known to those skilled inthe art who will readily appreciate that such probes may vary greatly inlength and may be labeled with a detectable label, such as aradioisotope or flourescent dye, to facilitate detection of the probe.DNA probe molecules may be produced by insertion of a DNA molecule whichencodes the polypeptides of this invention into suitable vectors, suchas plasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

[0138] RNA probes may be generated by inserting the DNA molecule whichencodes the polypeptides of this invention downstream of a bacteriophagepromoter such as T3, T7, or SP6. Large amounts of RNA probe may beproduced by incubating the labeled nucleotides with the linearizedfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

[0139] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to RNA encoding a humanMCH1 receptor, so as to prevent translation of the RNA. This inventionalso provides an antisense oligonucleotide having a sequence capable ofspecifically hybridizing to genomic DNA encoding a human MCH1 receptor.In one embodiment, the oligonucleotide comprises chemically modifiednucleotides or nucleotide analogues.

[0140] This invention provides an antibody capable of binding to a humanMCH1 receptor encoded by a nucleic acid encoding a human MCH1 receptor.This invention also provides an agent capable of competitivelyinhibiting the binding of the antibody to a human MCH1 receptor. In oneembodiment, the antibody is a monoclonal antibody or antisera.

[0141] This invention provides a pharmaceutical composition comprising(a) an amount of the oligonucleotide capable of passing through a cellmembrane and effective to reduce expression of a human MCH1 receptor and(b) a pharmaceutically acceptable carrier capable of passing through thecell membrane. In an embodiment, the oligonucleotide is coupled to asubstance which inactivates mRNA. In a further embodiment, the substancewhich inactivates mRNA is a ribozyme. In another embodiment, thepharmaceutically acceptable carrier comprises a structure which binds toa human MCH1 receptor on a cell capable of being taken up by the cellsafter binding to the structure. In a further embodiment, thepharmaceutically acceptable carrier is capable of binding to a humanMCH1 receptor which is specific for a selected cell type.

[0142] This invention provides a pharmaceutical composition whichcomprises an amount of an antibody effective to block binding of aligand to a human MCH1 receptor and a pharmaceutically acceptablecarrier.

[0143] As used herein, the phrase “pharmaceutically acceptable carrier”means any of the standard pharmaceutically acceptable carriers. Examplesinclude, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

[0144] This invention provides a transgenic, nonhuman mammal expressingDNA encoding a human MCH1 receptor. This invention also provides atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native human MCH1 receptor. This invention furtherprovides a transgenic, nonhuman mammal whose genome comprises antisenseDNA complementary to the DNA encoding a human MCH1 receptor so placedwithin the genome as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the human MCH1 receptor and whichhybridizes to mRNA encoding the human MCH1 receptor, thereby reducingits translation. In an embodiment, the DNA encoding the human MCH1receptor additionally comprises an inducible promoter. In anotherembodiment, the DNA encoding the human MCH1 receptor additionallycomprises tissue specific regulatory elements. In a further embodiment,the transgenic, nonhuman mammal is a mouse.

[0145] Animal model systems which elucidate the physiological andbehavioral roles of the polypeptides of this invention are produced bycreating transgenic animals in which the activity of the polypeptide iseither increased or decreased, or the amino acid sequence of theexpressed polypeptide is altered, by a variety of techniques. Examplesof these techniques include, but are not limited to: 1) Insertion ofnormal or mutant versions of DNA encoding the polypeptide, bymicroinjection, electroporation, retroviral transfection or other meanswell known to those in the art, into appropriate fertilized embryos inorder to produce a transgenic animal or 2) Homologous recombination ofmutant or normal, human or animal versions of these genes with thenative gene locus in transgenic animals to alter the regulation ofexpression or the structure of these polypeptide sequences. Thetechnique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and so is useful forproducing an animal that cannot express native polypeptides but doesexpress, for example, an inserted mutant polypeptide, which has replacedthe native polypeptide in the animal's genome by recombination,resulting in underexpression of the transporter. Microinjection addsgenes to the genome, but does not remove them, and so is useful forproducing an animal which expresses its own and added polypeptides,resulting in overexpression of the polypeptides.

[0146] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium such as M2 medium. DNA or cDNAencoding a polypeptide of this invention is purified from a vector bymethods well known in the art. Inducible promoters may be fused with thecoding region of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively, or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipette puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

[0147] This invention provides a process for identifying a chemicalcompound which specifically binds to a mammalian MCH1 receptor whichcomprises contacting cells comprising DNA encoding, and expressing ontheir cell surface, the mammalian MCH1 receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian MCH1 receptor, wherein the cells donot normally express the mammalian MCH1 receptor and the DNA encodingthe mammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (SEQ ID NO: 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. This invention also provides aprocess for identifying a chemical compound which specifically binds toa mammalian MCH1 receptor which comprises contacting a membranepreparation from cells comprising DNA encoding, and expressing on theircell surface, the mammalian MCH1 receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian MCH1 receptor, wherein the cells donot normally express the mammalian MCH1 receptor and the DNA encodingthe mammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (SEQ ID NO: 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. In one embodiment, the MCH1 receptoris a human MCH1 receptor. In another embodiment, the MCH1 receptor is arat MCH1 receptor. In another embodiment, the mammalian MCH1 receptorcomprises substantially the same amino acid sequence as the sequence ofthe human MCH1 receptor encoded by plasmid pEXJ.HR-TL231. In a furtherembodiment, the mammalian MCH1 receptor comprises substantially the sameamino acid sequence as that shown in FIG. 2 (SEQ ID NO: 2). In anotherembodiment, the mammalian MCH1 receptor comprises the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2). In a different embodiment, themammalian MCH1 receptor comprises the amino acid sequence shown in FIG.13 (SEQ ID NO: 27). In another embodiment, the mammalian MCH1 receptorcomprises the amino acid sequence shown in FIG. 14 (SEQ ID NO: 28). Instill another embodiment, the mammalian MCH1 receptor comprises theamino acid sequence shown in FIG. 15 (SEQ ID NO: 29). In one embodiment,the compound is not previously known to bind to a mammalian MCH1receptor. This invention further provides a compound identified by theabove-described processes.

[0148] In one embodiment of the above-described processes, the cell isan insect cell. In another embodiment, the cell is a mammalian cell. Ina further embodiment, the cell is nonneuronal in origin. In a furtherembodiment, the nonneuronal cell is a COS-7 cell, 293 human embryonickidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-)cell.

[0149] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian MCH1 receptor which comprises contacting cells expressing ontheir cell surface the mammalian MCH1 receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and separately with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting specific bindingof the chemical compound to the mammalian MCH1 receptor, a decrease inthe binding of the second chemical compound to the mammalian MCH1receptor in the presence of the chemical compound indicating that thechemical compound binds to the mammalian MCH1 receptor, wherein thecells do not normally express the mammalian MCH1 receptor and the DNAencoding the mammalian MCH1 receptor (a) hybridizes to a nucleic acidhaving the defined sequence shown in FIG. 1 (SEQ ID NO: 1) under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a MCH1 ligand is added to the culture and theCHO cells contain the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement.

[0150] This invention also provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian MCH1 receptor which comprises contacting a membranepreparation from cells expressing on their cell surface the mammalianMCH1 receptor, with both the chemical compound and a second chemicalcompound known to bind to the receptor, and separately with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian MCH1 receptor, a decrease in the binding of the secondchemical compound to the mammalian MCH1 receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian MCH1 receptor, wherein the cells do not normally express themammalian MCH1 receptor and the DNA encoding the mammalian MCH1 receptor(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 1 (SEQ ID NO: 1) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a MCH1 ligand isadded to the culture and the CHO cells contain the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.

[0151] In one embodiment, the mammalian MCH1 receptor is a human MCH1receptor or a mutant of such human MCH1 receptor which is activated byMCH or an analog or homolog thereof. In another embodiment, themammalian MCH1 receptor is a rat MCH1 receptor. In another embodiment,the mammalian MCH1 receptor comprises substantially the same amino acidsequence as the human MCH1 receptor encoded by plasmid pEXJ.HR-TL231. Ina further embodiment, the mammalian MCH1 receptor comprisessubstantially the same amino acid sequence as that shown in FIG. 2 (SEQID NO: 2). In another embodiment, the mammalian MCH1 receptor comprisesthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2).

[0152] In one embodiment, the cell is an insect cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, thecell is nonneuronal in origin. In another embodiment, the nonneuronalcell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, aNIH-3T3 cell, a mouse Y1 cell, or a LM(tk-) cell. In one embodiment, thecompound is not previously known to bind to a mammalian MCH1 receptor.

[0153] This invention provides a compound identified by theabove-described processes.

[0154] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian MCH1 receptor toidentify a compound which specifically binds to the mammalian MCH1receptor, which comprises (a) contacting cells transfected with andexpressing DNA encoding the mammalian MCH1 receptor with the pluralityof compounds not known to bind specifically to the mammalian MCH1receptor, under conditions permitting binding of compounds known to bindthe mammalian MCH1 receptor; (b) determining whether the binding of acompound known to bind to the mammalian MCH1 receptor is reduced in thepresence of the compounds within the plurality of compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so (c) separately determining the binding to themammalian MCH1 receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian MCH1 receptor.

[0155] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian MCH1 receptor toidentify a compound which specifically binds to the mammalian MCH1receptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing the mammalian MCH1 receptor withthe plurality of compounds not known to bind specifically to themammalian MCH1 receptor, under conditions permitting binding ofcompounds known to bind the mammalian MCH1 receptor; (b) determiningwhether the binding of a compound known to bind to the mammalian MCH1receptor is reduced in the presence of the compounds within theplurality of compounds, relative to the binding of the compound in theabsence of the plurality of compounds; and if so (c) separatelydetermining the binding to the mammalian MCH1 receptor of compoundsincluded in the plurality of compounds, so as to thereby identify thecompound which specifically binds to the mammalian MCH1 receptor.

[0156] In one embodiment of the above-described methods, the mammalianMCH1 receptor is a human MCH1 receptor or a mutant of such human MCH1receptor which is activated by MCH or an analog or homolog thereof. Inanother embodiment, the mammalian MCH1 receptor is a rat MCH1 receptor.In another embodiment, the cell is a mammalian cell. In a furtherembodiment, the mammalian cell is non-neuronal in origin. In anotherembodiment, the non-neuronal cell is a COS-7 cell, a 293 human embryonickidney cell, a LM(tk-) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3cell.

[0157] This invention also provides a method of detecting expression ofa mammalian MCH1 receptor by detecting the presence of mRNA coding forthe mammalian MCH1 receptor which comprises obtaining total mRNA fromthe cell and contacting the mRNA so obtained from a nucleic acid probeunder hybridizing conditions, detecting the presence of mRNA hybridizingto the probe, and thereby detecting the expression of the mammalian MCH1receptor by the cell.

[0158] This invention further provides a method of detecting thepresence of a mammalian MCH1 receptor on the surface of a cell whichcomprises contacting the cell with an antibody under conditionspermitting binding of the antibody to the receptor, detecting thepresence of the antibody bound to the cell, and thereby detecting thepresence of the mammalian MCH1 receptor on the surface of the cell.

[0159] This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a transgenic, nonhuman mammal whose levels of humanMCH1 receptor activity are varied by use of an inducible promoter whichregulates human MCH1 receptor expression.

[0160] This invention also provides a method of determining thephysiological effects of varying levels of activity of human MCH1receptors which comprises producing a panel of transgenic, nonhumanmammals each expressing a different amount of human MCH1 receptor.

[0161] This invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a human MCH1 receptorcomprising administering a compound to a transgenic, nonhuman mammal,and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal asa result of overactivity of a human MCH1 receptor, the alleviation ofthe abnormality identifying the compound as an antagonist. Thisinvention also provides an antagonist identified by the above-describedmethod. This invention further provides a pharmaceutical compositioncomprising an antagonist identified by the above-described method and apharmaceutically acceptable carrier. This invention provides a method oftreating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a human MCH1 receptor whichcomprises administering to the subject an effective amount of thispharmaceutical composition, thereby treating the abnormality.

[0162] This invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a human MCH1receptor comprising administering a compound to transgenic, nonhumanmammal, and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by theabove-described method. This invention further provides a pharmaceuticalcomposition comprising an agonist identified by the above-describedmethod and a pharmaceutically acceptable carrier. This invention furtherprovides a method of treating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a human MCH1receptor which comprises administering to the subject an effectiveamount of this pharmaceutical composition, thereby treating theabnormality.

[0163] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific mammalianallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human MCH1 receptor and labeled with a detectable marker; (e)detecting labeled bands which have hybridized to the DNA encoding ahuman MCH1 receptor labeled with a detectable marker to create a uniqueband pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same. In one embodiment, a disorder associated withthe activity of a specific mammalian allele is diagnosed.

[0164] This invention provides a method of preparing the purified humanMCH1 receptor which comprises: (a) inducing cells to express the humanMCH1 receptor; (b) recovering the human MCH1 receptor from the inducedcells; and (c) purifying the human MCH1 receptor so recovered.

[0165] This invention provides a method of preparing the purified humanMCH1 receptor which comprises: (a) inserting nucleic acid encoding thehuman MCH1 receptor in a suitable vector; (b) introducing the resultingvector in a suitable host cell; (c) placing the resulting cell insuitable condition permitting the production of the isolated human MCH1receptor; (d) recovering the human MCH1 receptor produced by theresulting cell; and (e) purifying the human MCH1 receptor so recovered.

[0166] This invention provides a process for determining whether achemical compound is a mammalian MCH1 receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian MCH1 receptor with the compound under conditions permittingthe activation of the mammalian MCH1 receptor, and detecting an increasein mammalian MCH1 receptor activity, so as to thereby determine whetherthe compound is a mammalian MCH1 receptor agonist. This invention alsoprovides a process for determining whether a chemical compound is amammalian MCH1 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the mammalian MCH1 receptorwith the compound in the presence of a known mammalian MCH1 receptoragonist, under conditions permitting the activation of the mammalianMCH1 receptor, and detecting a decrease in mammalian MCH1 receptoractivity, so as to thereby determine whether the compound is a mammalianMCH1 receptor antagonist. In one embodiment, the mammalian MCH1 receptoris a human MCH1 receptor or a mutant of such human MCH1 receptor whichis activated by MCH or an analog or homolog thereof.

[0167] This invention further provides a pharmaceutical compositionwhich comprises an amount of a mammalian MCH1 receptor agonistdetermined by the above-described process effective to increase activityof a mammalian MCH1 receptor and a pharmaceutically acceptable carrier.In one embodiment, the mammalian MCH1 l receptor agonist is notpreviously known.

[0168] This invention provides a pharmaceutical composition whichcomprises an amount of a mammalian MCH1 receptor antagonist determinedby the above-described process effective to reduce activity of amammalian MCH1 receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian MCH1 receptor antagonist is not previouslyknown.

[0169] This invention provides a process for determining whether achemical compound specifically binds to and activates a mammalian MCH1receptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the mammalian MCH1receptor, wherein such cells do not normally express the mammalian MCH1receptor, with the chemical compound under conditions suitable foractivation of the mammalian MCH1 receptor, and measuring the secondmessenger response in the presence and in the absence of the chemicalcompound, a change in the second messenger response in the presence ofthe chemical compound indicating that the compound activates themammalian MCH1 receptor. In one embodiment, the second messengerresponse comprises chloride channel activation and the change in secondmessenger is an increase in the level of inward chloride current.

[0170] This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian MCH1 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian MCH1 receptor, wherein such cells do not normallyexpress the mammalian MCH1 receptor, with both the chemical compound anda second chemical compound known to activate the mammalian MCH1receptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian MCH1 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian MCH1 receptor. In one embodiment, the secondmessenger response comprises chloride channel activation and the changein second messenger response is a smaller increase in the level ofinward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound. This invention also provides the above-describedprocesses performed with membrane preparations from cells producing asecond messenger response and transfected with and expressing themammalian MCH1 receptor.

[0171] In one embodiment of the above-described processes, the mammalianMCH1 receptor is a human MCH1 receptor or a mutant of such human MCH1receptor which is activated by MCH or an analog or homolog thereof. Inanother embodiment, the mammalian MCH1 receptor is a rat MCH1 receptor.In another embodiment, the mammalian MCH1 receptor comprisessubstantially the same amino acid sequence as encoded by the plasmidpEXJ.HR-TL231. In a further embodiment, the mammalian MCH1 receptorcomprises substantially the same amino acid sequence as that shown inFIG. 2 (SEQ ID NO: 2). In another embodiment, the mammalian MCH1receptor comprises an amino acid sequence as shown in FIG. 2 (SEQ ID NO:2). In an embodiment, the cell is an insect cell. In a furtherembodiment, the cell is a mammalian cell. In a still further embodiment,the mammalian cell is nonneuronal in origin. In another embodiment, thenonneuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidneycell, NIH-3T3 cell or LM(tk-) cell. In an embodiment, the compound isnot previously known to bind to a mammalian MCH1 receptor. Thisinvention also provides a compound determined by the above-describedprocesses.

[0172] This invention also provides a pharmaceutical composition whichcomprises an amount of a mammalian MCH1 receptor agonist determined bythe above-described processes effective to increase activity of amammalian MCH1 receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian MCH1 receptor agonist is not previouslyknown.

[0173] This invention further provides a pharmaceutical compositionwhich comprises an amount of a mammalian MCH1 receptor antagonistdetermined by the above-described processes effective to reduce activityof a mammalian MCH1 receptor and a pharmaceutically acceptable carrier.In one embodiment, the mammalian MCH1 receptor antagonist is notpreviously known.

[0174] This invention provides a method of screening a plurality ofchemical compounds not known to activate a mammalian MCH1 receptor toidentify a compound which activates the mammalian MCH1 receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian MCH1 receptor with the plurality of compounds not known toactivate the mammalian MCH1 receptor, under conditions permittingactivation of the mammalian MCH1 receptor; (b) determining whether theactivity of the mammalian MCH1 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the mammalian MCH1 receptor is increased by each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which activates the mammalian MCH1 receptor. In one embodiment,the mammalian MCH1 receptor is a human MCH1 receptor or a mutant of suchhuman MCH1 receptor which is activated by MCH or an analog or homologthereof. In another embodiment, the mammalian MCH1 receptor is a ratMCH1 receptor.

[0175] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a mammalianMCH1 receptor to identify a compound which inhibits the activation ofthe mammalian MCH1 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian MCH1 receptor with theplurality of compounds in the presence of a known mammalian MCH1receptor agonist, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activation of themammalian MCH1 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian MCH1 receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian MCH1 receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian MCH1 receptor. In one embodiment, the mammalian MCH1 receptoris a human MCH1 receptor or a mutant of such human MCH1 receptor whichis activated by MCH or an analog or homolog thereof. In anotherembodiment, the mammalian MCH1 receptor is a rat MCH1 receptor.

[0176] In one embodiment of the above-described methods, the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or anNIH-3T3 cell.

[0177] This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to increasemammalian MCH1 receptor activity and a pharmaceutically acceptablecarrier.

[0178] This invention also provides a pharmaceutical compositioncomprising a compound identified by the above-described methodseffective to decrease mammalian MCH1 receptor activity and apharmaceutically acceptable carrier.

[0179] This invention further provides a method of measuring receptoractivation in an oocyte expression system such as a Xenopus oocyteexpression system or melanophore. In an embodiment, receptor activationis determined by measurement of ion channel activity. In anotherembodiment, receptor activation is measured by aequorin luminescence.

[0180] Expression of genes in Xenopus oocytes is well known in the art(Coleman, A., 1984; Masu, Y.,et al., 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (Sambrook, et al. 1989) including usingT7 polymerase with the mCAP RNA mapping kit (Stratagene).

[0181] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a mammalian MCH1 receptor which comprises administering to thesubject an amount of a compound which is a mammalian MCH1 receptoragonist effective to treat the abnormality. In separate embodiments, theabnormality is a regulation of a steroid or pituitary hormone disorder,an epinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder such as Alzheimer's disease, a sensory modulation andtransmission disorder, a motor coordination disorder, a sensoryintegration disorder, a motor integration disorder, a dopaminergicfunction disorder such as Parkinson's disease, a sensory transmissiondisorder, an olfaction disorder, a sympathetic innervation disorder, anaffective disorder such as depression, a stress-related disorder, afluid-balance disorder, a urinary disorder such as urinary incontinence,a seizure disorder, pain, psychotic behavior such as schizophrenia,morphine tolerance, opiate addiction or migraine.

[0182] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a mammalian MCH1 receptor which comprises administering to thesubject an amount of a compound which is a mammalian MCH1 receptorantagonist effective to treat the abnormality. In separate embodiments,the abnormality is a regulation of a steroid or pituitary hormonedisorder, an epinephrine release disorder, a gastrointestinal disorder,a cardiovascular disorder, an electrolyte balance disorder,hypertension, diabetes, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder such as Alzheimer's disease, a sensorymodulation and transmission disorder, a motor coordination disorder, asensory integration disorder, a motor integration disorder, adopaminergic function disorder such as Parkinson's disease, a sensorytransmission disorder, an olfaction disorder, a sympathetic innervationdisorder, an affective disorder such as depression, a stress-relateddisorder, a fluid-balance disorder, a urinary disorder such as urinaryincontinence, a seizure disorder, pain, psychotic behavior such asschizophrenia, morphine tolerance, opiate addiction or migraine.

[0183] This invention provides a process for making a composition ofmatter which specifically binds to a mammalian MCH1 receptor whichcomprises identifying a chemical compound using any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian MCH1 receptor and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof. In one embodiment, the mammalian MCH1receptor is a human MCH1 receptor or a mutant of such human MCH1receptor which is activated by MCH or an analog or homolog thereof. Inanother embodiment, the mammalian MCH1 receptor is a rat MCH1 receptor.

[0184] This invention further provides a process for preparing acomposition which comprises admixing a pharmaceutically acceptablecarrier and a therapeutically effective amount of a chemical compoundidentified by any of the processes described herein for identifying acompound which binds to and/or activates or inhibits activation of amammalian MCH1 receptor or a novel structural and functional analog orhomolog thereof. In one embodiment, the mammalian MCH1 receptor is ahuman MCH1 receptor or a mutant of such human MCH1 receptor which isactivated by MCH or an analog or homolog thereof. In another embodiment,the mammalian MCH1 receptor is a rat MCH1 receptor.

[0185] This invention provides a process for determining whether achemical compound is a human MCH1 receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding the humanMCH1 receptor with the compound in the presence of a known human MCH1receptor agonist, under conditions permitting the activation of thehuman MCH1 receptor, and detecting a decrease in human MCH1 receptoractivity, so as to thereby determine whether the compound is a humanMCH1 receptor antagonist, wherein the DNA encoding the human MCH1receptor comprises the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH,and the cells do not express the MCH1 receptor prior to transfectingthem.

[0186] This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of ahuman MCH1 receptor, which comprises separately contacting cellsexpressing on their cell surface the human MCH1 receptor and producing asecond messenger response upon activation of the human MCH1 receptor,wherein such cells do not normally express the human MCH1 receptor andthe DNA encoding the human MCH1 receptor comprises the sequence shown inFIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197), with both the chemical compound and a secondchemical compound known to activate the human MCH1 receptor, and withonly the second chemical compound, under conditions suitable foractivation of the human MCH1 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of the humanMCH1 receptor, wherein the second chemical compound is MCH or a homologor analog of MCH. In one embodiment, the second messenger responsecomprises chloride channel activation and the change in second messengerresponse is a smaller increase in the level of inward chloride currentin the presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compound.

[0187] This invention further provides a method of screening a pluralityof chemical compounds not known to inhibit the activation of a humanMCH1 receptor to identify a compound which inhibits the activation ofthe human MCH1 receptor, which comprises:

[0188] (a) contacting cells transfected with and expressing the humanMCH1 receptor, wherein such cells do not normally express the human MCH1receptor and the DNA encoding the human MCH1 receptor comprises thesequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), with the plurality ofcompounds in the presence of a known human MCH1 receptor agonist, underconditions permitting activation of the human MCH1 receptor, wherein theknown MCH1 receptor agonist is MCH or a homolog or analog of MCH;

[0189] (b) determining whether the activation of the human MCH1 receptoris reduced in the presence of the plurality of compounds, relative tothe activation of the human MCH1 receptor in the absence of theplurality of compounds; and if so

[0190] (c) separately determining the extent of inhibition of activationof the human MCH1 receptor for each compound included in the pluralityof compounds, so as to thereby identify the compound which inhibits theactivation of the human MCH1 receptor.

[0191] In one embodiment of the above-described methods, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. Instill another embodiment, the cell is a mammalian cell which isnonneuronal in origin. In further embodiments, the cell is a COS-7 cell,a CHO cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a mouseY1 cell, or a LM(tk-) cell.

[0192] This invention provides a process for making a composition ofmatter which specifically binds to a human MCH1 receptor which comprisesidentifying a chemical compound which specifically binds to the humanMCH1 receptor and then synthesizing the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting cellsexpressing on their cell surface the human MCH1 receptor, with both thechemical compound and a second chemical compound known to bind to thereceptor, and separately with only the second chemical compound, underconditions suitable for binding of both compounds, and detecting theextent of specific binding of the chemical compound to the human MCH1receptor, a decrease in the binding of the second chemical compound tothe human MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the human MCH1 receptor,wherein the cells do not normally express the human MCH1 receptor, thehuman MCH1 receptor is encoded by nucleic acid comprising the sequenceshown in FIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231(ATCC Accession No. 203197), and the second chemical compound is MCH ora homolog or analog of MCH.

[0193] This invention further provides a process for making acomposition of matter which specifically binds to a human MCH1 receptorwhich comprises identifying a chemical compound which specifically bindsto the human MCH1 receptor and then synthesizing the chemical compoundor a structural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting amembrane preparation from cells expressing on their cell surface thehuman MCH1 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and separately withonly the second chemical compound, under conditions suitable for bindingof both compounds, and detecting the extent of specific binding of thechemical compound to the human MCH1 receptor, a decrease in the bindingof the second chemical compound to the human MCH1 receptor in thepresence of the chemical compound indicating that the chemical compoundbinds to the human MCH1 receptor, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and thesecond chemical compound is MCH or a homolog or analog of MCH.

[0194] This invention also provides a process for making a compositionof matter which is a human MCH1 receptor antagonist which comprisesidentifying a chemical compound which is a human MCH1 receptorantagonist and then synthesizing the chemical compound or a structuraland functional analog or homolog thereof, wherein the chemical compoundis identified as a human MCH1 receptor antagonist by a process whichcomprises contacting cells transfected with and expressing DNA encodingthe human MCH1 receptor with the compound in the presence of a knownhuman MCH1 receptor agonist, under conditions permitting the activationof the human MCH1 receptor, and detecting a decrease in human MCH1receptor activity, so as to thereby determine whether the compound is ahuman MCH1 receptor antagonist, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH.

[0195] This invention still further provides a process for making acomposition of matter which specifically binds to and inhibits theactivation of a human MCH1 receptor which comprises identifying achemical compound which specifically binds to and inhibits theactivation of the human MCH1 receptor and then synthesizing the chemicalcompound or a structural and functional analog or homolog thereof,wherein the chemical compound is identified as binding to and inhibitingthe activation of the human MCH1 receptor by a process which comprisesseparately contacting cells expressing on their cell surface the humanMCH1 receptor and producing a second messenger response upon activationof the human MCH1 receptor, wherein such cells do not normally expressthe human MCH1 receptor and the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), withboth the chemical compound and a second chemical compound known toactivate the human MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the human MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the human MCH1 receptor, wherein the second chemicalcompound is MCH or a homolog or analog of MCH. In one embodiment, thesecond messenger response comprises chloride channel activation and thechange in second messenger response is a smaller increase in the levelof inward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound.

[0196] This invention provides a process for preparing a compositionwhich comprises identifying a chemical compound which specifically bindsto a human MCH1 receptor, and then admixing a carrier and the chemicalcompound or a structural and functional analog or homolog thereof,wherein the chemical compound is identified as binding to the human MCH1receptor by a process involving competitive binding which comprisescontacting cells expressing on their cell surface the human MCH1receptor, with both the chemical compound and a second chemical compoundknown to bind to the receptor, and separately with only the secondchemical compound, under conditions suitable for binding of bothcompounds, and detecting the extent of specific binding of the chemicalcompound to the human MCH1 receptor, a decrease in the binding of thesecond chemical compound to the human MCH1 receptor in the presence ofthe chemical compound indicating that the chemical compound binds to thehuman MCH1 receptor, wherein the cells do not normally express the humanMCH1 receptor, the human MCH1 receptor is encoded by nucleic acidcomprising the sequence shown in FIG. 1 (Seq. ID No. 1) or contained inplasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and the secondchemical compound is MCH or a homolog or analog of MCH.

[0197] This invention further provides a process for preparing acomposition which comprises identifying a chemical compound whichspecifically binds to a human MCH1 receptor, and then admixing a carrierand the chemical compound or a structural and functional analog orhomolog thereof, wherein the chemical compound is identified as bindingto the human MCH1 receptor by a process involving competitive bindingwhich comprises contacting a membrane preparation from cells expressingon their cell surface the human MCH1 receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and separately with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting the extent ofspecific binding of the chemical compound to the human MCH1 receptor, adecrease in the binding of the second chemical compound to the humanMCH1 receptor in the presence of the chemical compound indicating thatthe chemical compound binds to the human MCH1 receptor, wherein thecells do not normally express the human MCH1 receptor, the human MCH1receptor is encoded by nucleic acid comprising the sequence shown inFIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197), and the second chemical compound is MCH or ahomolog or analog of MCH.

[0198] This invention also provides a process for preparing acomposition which comprises identifying a chemical compound which is ahuman MCH1 receptor antagonist, and then admixing a carrier and thechemical compound or a structural and functional analog or homologthereof, wherein the chemical compound is identified as a human MCH1receptor antagonist by a process which comprises contacting cellstransfected with and expressing DNA encoding the human MCH1 receptorwith the compound in the presence of a known human MCH1 receptoragonist, under conditions permitting the activation of the human MCH1receptor, and detecting a decrease in human MCH1 receptor activity, soas to thereby determine whether the compound is a human MCH1 receptorantagonist, wherein the cells do not normally express the human MCH1receptor, the human MCH1 receptor is encoded by nucleic acid comprisingthe sequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), and the known human MCH1receptor agonist is MCH or a homolog or analog of MCH.

[0199] This invention still further provides a process for preparing acomposition which comprises identifying a chemical compound whichspecifically binds to and inhibits the activation of a human MCH1receptor, and then admixing a carrier and the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to and inhibiting activationof the human MCH1 receptor by a process which comprises separatelycontacting cells expressing on their cell surface the human MCH1receptor and producing a second messenger response upon activation ofthe human MCH1 receptor, wherein such cells do not normally express thehuman MCH1 receptor and the human MCH1 receptor is encoded by nucleicacid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), withboth the chemical compound and a second chemical compound known toactivate the human MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the human MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the human MCH1 receptor, wherein the second chemicalcompound is MCH or a homolog or analog of MCH. In one embodiment, thesecond messenger response comprises chloride channel activation and thechange in second messenger response is a smaller increase in the levelof inward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound.

[0200] In one embodiment of any of the above methods, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. Inanother embodiment, the mammalian cell is nonneuronal in origin. Infurther embodiments, the nonneuronal cell is a COS-7 cell, a 293 humanembryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or aLM(tk-) cell.

[0201] Thus, once the gene for a targeted receptor subtype is cloned, itis placed into a recipient cell which then expresses the targetedreceptor subtype on its surface. This cell, which expresses a singlepopulation of the targeted human receptor subtype, is then propagatedresulting in the establishment of a cell line. This cell line, whichconstitutes a drug discovery system, is used in two different types ofassays: binding assays and functional assays. In binding assays, theaffinity of a compound for both the receptor subtype that is the targetof a particular drug discovery program and other receptor subtypes thatcould be associated with side effects are measured. These measurementsenable one to predict the potency of a compound, as well as the degreeof selectivity that the compound has for the targeted receptor subtypeover other receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

[0202] Approaches to designing and synthesizing receptorsubtype-selective compounds are well known-and include traditionalmedicinal chemistry and the newer technology of combinatorial chemistry,both of which are supported by computer-assisted molecular modeling.With such approaches, chemists and pharmacologists use their knowledgeof the structures of the targeted receptor subtype and compoundsdetermined to bind and/or activate or inhibit activation of the receptorsubtype to design and synthesize structures that will have activity atthese receptor subtypes.

[0203] Combinatorial chemistry involves automated synthesis of a varietyof novel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

[0204] Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

[0205] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0206] Experimental Details

[0207] Materials and Methods

[0208] Cloning of Human MCH1 Receptor

[0209] Discovery of an Expressed Sequence Tac (EST) F07228 in GENEMBLHomologous to FB41a

[0210] A BLAST search of GENEMBL was performed with the GCG sequenceanalysis package (Genetics Computer Group, Madison, Wis.) using aSynaptic Pharmaceutical Corporation proprietary sequence, FB41a, as aquery. This resulted in the identification of an EST (accession numberF07228) with a high degree of homology to FB41a and somatostatin, opiateand galanin receptors.

[0211] Construction and Screening of a Human Hippocampal cDNA Library

[0212] Poly A+ RNA was purified from human hippocampal RNA (Clontech)using a FastTrack kit (Invitrogen, Corp.). DS-cDNA was synthesized frompoly A+ RNA according to Gubler and Hoffman (1983) with minormodifications. The resulting cDNA was ligated to BstXI adaptors.(Invitrogen, Corp.) and the excess adaptors removed by exclusion columnchromatography. High molecular weight fractions of size-selected ds-cDNAwere ligated in pEXJ.BS, an Okayama and Berg expression vector modifiedfrom pcEXV (Miller and Germain, 1986) to contain BstXI and otheradditional restriction sites. A total of 2.2×10⁶ independent clones witha mean insert size of 3.0 kb were generated. The library was plated onagar plates (ampicillin selection) and glycerol stocks for 450 pools of5000 independent clones were prepared. Primary glycerol stocks were alsogrouped together in groups of approximately 10 to create superpools.

[0213] Cloning of the Full-Length Sequence of MCH1

[0214] Glycerol stocks of the superpools and primary pools from thehuman hippocampal cDNA library were screened by PCR with F07228 specificprimers T579 and T580 using Taq DNA Polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and the following PCR protocol: 94° C. hold for 5minutes; 40 cycles of 94° C. for 2 minute, 68° C. for 4 minutes; 7minute hold at 68° C.; 4° C. hold until the samples are run on a gel.One positive primary pool 490, was successively divided into subpools,amplified in LB medium overnight and screened by PCR using primers T579and T580. One positive subpool, 490-4-10-23 was plated on agar plates(ampicillin selection), and colonies were transferred to nitrocellulosemembranes (Schleicher and Schuell, Keene, N.H.). Filters were hybridizedfor two days under high stringency conditions with 10⁶ cpm/ml of a³²P-labeled cDNA probe, T581, designed against the F07228 EST sequence.Filters were washed and apposed to Biomax MS film (Kodak). Sevenpositive colonies were picked, streaked on LB-AMP plates, and grownovernight. Two individual colonies from each of the original seven werepicked and subjected to vector-anchored PCR using the following primerpairs: T95, T580 and T94, T579. One positive colony, G1, was amplifiedovernight in TB and processed for plasmid purification. This plasmid wasdesignated TL230 and sequenced on both strands with a Sequenase kit (USBiochemical, Cleveland, Ohio). Nucleotide and peptide sequence analysiswere performed with GCG programs (Genetics Computer Group, Madison,Wis.). A HindIII-KpnI fragment of TL230 was subcloned into the mammalianexpression vector pEXJ, and named TL231.

[0215] Primers and Probes: TL579: 5′-GGGAACTCCACGGTCATCTTCGC (SEQ ID NO:5) GGT-3′ TL580: 5′-TAGCGGTCAATGGCCATGGCGGT (SEQ ID NO: 6) CAG-3′ TL581:5′-CTCCTGGGCATGCCCTTCATGATCCA (SEQ ID NO: 7) CCAGCTCATGGGCAATGGG-3′TL94: 5′-CTTCTAGGCCTGTACGGAAGTG (SEQ ID NO: 8) TTA-3′ TL95:5′-GTTGTGGTTTGTCCAAACTCATCA (SEQ ID NO: 9) ATG-3′

[0216] Isolation of a Fragment of a Species Homologue of TL231 (HumanMCH1)

[0217] To obtain a fragment of a species homologue of TL231, the speciesgenomic DNA (Clontech) may be amplified with a forward PCR primercorresponding to one of the TM regions of TL231 and a reverse primercorresponding to another TM region of TL231. PCR may be performed withthe Expand Long Template PCR System (Boeringer Mannheim), for example,under the following conditions: 30 sec at 94° C., 1.5 min at 50 ° C.,1.5 min at 68° C. for 40 cycles, with a pre- and post-incubation of 5min at 94° C. and 7 min at 68° C., respectively. A band is isolated,subcloned using the TA cloning kit (Invitrogen), and sequenced. Thesequence is run and analyzed on an ABI PRISM 377 BigDye Terminator CycleSequencing Kit Sequencer. Forward and reverse PCR primers are designedagainst this sequence and used to amplify a band from genomic DNA using,for example, the following conditions: 30 sec at 94° C., 1.5 min at 68°C. for 35 cycles, with a pre- and post-incubation of 5 min at 94° C. and5 min at 68° C., respectively. The PCR product is subcloned using the TAcloning kit (Invitrogen). Miniprep cultures of transformants areprepared and sequenced as above.

[0218] Isolation of a Full-Length Species Homolog of TL231 (Human MCH1)

[0219] A nucleic acid sequence encoding an MCH1 receptor may be isolatedusing standard molecular biology techniques and approaches such as thosebriefly described below:

[0220] Approach #1: To obtain a full-length MCH1 receptor, a cosmidlibrary could be screened with a ³²P-labeled oligonucleotide probe.

[0221] The full-length sequence may be obtained by sequencing thiscosmid clone with additional sequencing primers. Since one intron ispresent in this gene the full-length intronless gene may be obtainedfrom cDNA using standard molecular biology techniques. For example, aforward PCR primer designed in the 5′UT and a reverse PCR primerdesigned in the 3′UT may be used to amplify a full-length, intronlessgene from cDNA. Standard molecular biology techniques could be used tosubclone this gene into a mammalian expression vector.

[0222] Approach #2: Standard molecular biology techniques could be usedto screen commercial cDNA phage libraries by hybridization under highstringency with a ³²P-labeled oligonucleotide probe. One may isolate afull-length MCH1 receptor by obtaining a plaque purified clone from thelambda libraries and then subjecting the clone to direct DNA sequencing.Alternatively, standard molecular biology techniques could be used toscreen in-house cDNA plasmid libraries by PCR amplification of librarypools using primers to the MCH1 sequence. A full-length clone could beisolated by Southern hybridization of colony lifts of positive poolswith a ³²P-labeled oligonucleotide probe.

[0223] Approach #3: As yet another alternative method, one could utilize3′ and 5′ RACE to generate PCR products from cDNA expressing MCH1 whichcontain the additional sequences of MCH1. These RACE PCR products couldthen be sequenced to determine the missing sequence. This new sequencecould then be used to design a forward PCR primer in the 5′UT and areverse primer in the 3′UT. These primers could then be used to amplifya full-length MCH1 clone from cDNA.

[0224] Construction of Human MCH1 Mutants

[0225] The plasmid TL231 encodes three in frame methionine residues, anyof which could potentially initiate translation of the MCH1 receptor.The ability of these residues to function in a heterologous expressionsystem was examined by constructing mutants of TL231 in which one ormore of the downstream methionine residues was mutated to alanine.Mutagenesis was performed using the QuickChange site-directedmutagenesis kit (Stratagene). Each 50 ul PCR reaction contained 10 mMKCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, 0.1% TritonX-100, 0.1 mg/ml nuclease-free BSA, 114 ng each of two mutagenesisprimers (see below), 50 ng of plasmid DNA template (see below), 2.5units of PfuTurbo DNA polymerase, and 1 ul of the proprietary dNTP mixprovided in the kit. Thermocycling was performed with an AppliedBiosystems 9700 machine using the following cycling parameters: onecycle of 95° for 30 seconds; eighteen cycles of 95° for 30 seconds, 55°for 1 minute, 68° for 2.5 minutes; a final hold at 4°. Next, 1 ul (10units) of DpnI restriction enzyme was added to the mutagenesis reactionfollowed by incubation at 37° for 1 hour. A 2 ul aliquot of thisdigestion was used to transform 50 ul of E.coli XL1-Blue cells providedwith the mutagenesis kit. Transformants were selected by their abilityto grow at 37° on LB plates containing 100 ug/ml ampicillin. Singlecolonies which resulted from the overnight incubation of the plates wereused to inoculate 2 ml cultures of LB-ampicillin and allowed to growovernight at 37° with shaking. Miniprep DNA was prepared from thesecultures using the Qiagen miniprep system and subjected to automatedsequence analysis. This allowed both the confirmation of the desiredmutation and the integrity of the remainder of the MCH1 coding sequence.After identification of a correctly mutated clone, a large scale DNAprep was prepared using a Qiagen megaprep column.

[0226] To create the clone encoding only the M70A mutation, the templateDNA was TL231 and the mutagenesis primers were RP192 and RP193. Thisclone is designated R106 (SEQ ID NO: 16) and encodes only the first twopotential start codons (See FIG. 12). To create the clone encoding boththe M6A and the M70A mutations, the template DNA was R106 and themutagenesis primers were RP190 and RP191. The resulting clone isdesignated R114 (SEQ ID NO: 17) and encodes only first start codon (SeeFIG. 12).

[0227] If desired, the same mutagenesis technology can be employed toconstruct additional MCH1 mutants that encode other combinations of theavailable methionine residues. The mutation M1A could be constructedusing primers X1 and X2. Such a change would eliminate the firstmethionine but retain the two downstream residues. Likewise, the doublemutation M1A, M70A could be constructed by sequentially using primerpairs X1/X2 and RP192/RP193. This would create a gene in which only thesecond methionine was left intact.

[0228] Primers used in the generation of hMCH1 mutant receptorconstructs: Mutant Primer Primer Sequence R106 RP1925′ CGGCACTGGCTGGGCGGACCTGGAAGCCTCG 3′ (SEQ ID NO: 18) M70A) RP1935′ CGAGGCTTCCAGGTCCGCCCAGCCAGTGCCG 3′ (SEQ ID NO: 19) R114 RP1905′ ATGTCAGTGGGAGCCGCGAAGAAGGGAGTGGG 3′ (SEQ ID NO: 20) (M6A, RP1915′ CCCACTCCCTTCTTCGCGGCTCCCACTGACAT 3′ (SEQ ID NO: 21) M70A) (M1A) X15′ TAATGTGTCTAGGTGGCGTCAGTGGGAGCCATG 3′ (SEQ ID NO: 22) X25′ CATGGCTCCCACTGACGCCACCTAGACACATTA 3′ (SEQ ID NO: 23)

[0229] Construction of a Short Form of the Human MCH1 Receptor

[0230] A short form of the human MCH1 receptor expressing only the mostdownstream of the three potential initiating methionines was generatedas follows. TL231 was amplified with BB1122 (a forward primer beginning10 nucleotides upstream of the third methionine in TL231, and alsoincorporating a HindIII site) and BB1123 (a reverse primer in the secondtransmembrane domain) and the resulting product digested with HindIIIand BgIIIA. PCR was performed with the Expand Long Template PCR System(Roche Molecular Biochemicals, Indianapolis, Ind.) under the followingconditions: 20 seconds at 94° C., 1 minute at 68° C. for 40 cycles, witha pre- and post-incubation of 5 minutes at 94° C. and 7 minutes at 68°C. respectively. The 270 bp product was gel purified and ligated to a 4kb HindIII/BgIII restriction fragment from TL231. The resultingconstruct was named B0120 (SEQ ID NO: 24).

[0231] Primers used in the construction of the truncated human MCH1receptor: BB1122 5′-TGACACTAAGCTTCACTGGCTGGA (SEQ ID NO: 25)TGGACCTGGAAGC-3′ BB1123 5′-GCCCAGGAGAAAGAGGAGATC (SEQ ID NO: 26) TAC-3′

[0232] Host Cells

[0233] A broad variety of host cells can be used to study heterologouslyexpressed proteins. These cells include but are not restricted toassorted mammalian lines such as; Cos-7, CHO, LM(tk-), HEK293, etc.;insect cell lines such as; Sf9, Sf21, etc.; amphibian cells such asxenopus oocytes; and others.

[0234] COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

[0235] Human embryonic kidney 293 cells are grown on 150 mm plates inDMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stockplates of 293 cells are trypsinized and split 1:6 every 3-4 days.

[0236] Mouse fibroblast LM(tk-) cells are grown on 150 mm plates inD-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk-) cells aretrypsinized and split 1:10 every 3-4 days.

[0237] Chinese hamster ovary (CHO) cells were grown on 150 mm plates inHAM's F-12 medium with supplements (10% bovine calf serum, 4 mML-glutamine and 100 units/ml penicillin/ 100 μg/ml streptomycin) at 37°C., 5% CO₂. Stock plates of CHO cells are trypsinized and split 1:8every 3-4 days.

[0238] Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mmplates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10%bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days.

[0239] Sf9 and Sf21 cells are grown in monolayers on 150 mm tissueculture dishes in TMN-FH media supplemented with 10% fetal calf serum,at 27° C., no CO₂. High Five insect cells are grown on 150 mm tissueculture dishes in Ex-Cell 400^(TH) medium supplemented with L-Glutamine,also at 27° C., no CO₂.

[0240] In some cases, cell lines that grow as adherent monolayers can beconverted to suspension culture to increase cell yield and provide largebatches of uniform assay material for routine receptor screeningprojects.

[0241] Xenopus oocytes can also be used as a host system for transientexpression of heterologous proteins. Their maintenance and usage isdescribed in the electrophysiological methods section that follows.

[0242] Transient Expression

[0243] DNA encoding proteins to be studied can be transiently expressedin a variety of mammalian, insect, amphibian and other cell lines byseveral methods including but not restricted to; calciumphosphate-mediated, DEAE-dextran mediated, Liposomal-mediated,viral-mediated, electroporation-mediated and microinjection delivery.Each of these methods may require optimization of assorted experimentalparameters depending on the DNA, cell line, and the type of assay to besubsequently employed.

[0244] A typical protocol for the calcium phosphate method as applied toLM(tk-) cells is described as follows; Adherent cells are harvestedapproximately twenty-four hours before transfection and replated at adensity of 1-2×10⁵ cells/cm² in a 100 mm tissue culture dish and allowedto incubate over night at 37° C. at 5% CO₂. 250 μl of a mixture of CaCl₂and DNA (20 μg DNA in 250 mM CaCl₂) is added to a 5 ml plastic tube and250 ul of 2×HBS (250 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, 12 mM dextrose,50 mM HEPES) is slowly added with gentle mixing. The mixture is allowedto incubate for 20 minutes at room temperature to allow a DNAprecipitate to form. The cells are then washed with complete medium, 10ml of culture medium is added to each plate, followed by addition of theDNA precipitate. The cells are then incubated for 24 to 48 hours at 37°C. at 5% CO₂.

[0245] A typical protocol for the DEAE-dextran method as applied toCos-7 cells is described as follows; Cells to be used for transfectionare split 24 hours prior to the transfection to provide flasks which are70-80% confluent at the time of transfection. Briefly, 8 pg of receptorDNA plus 8 μg of any additional DNA needed (e.g. G_(o) proteinexpression vector, reporter construct, antibiotic resistance marker,mock vector, etc.) are added to 9 ml of complete DMEM plus DEAE-dextranmixture (10 mg/ml in PBS). Cos-7 cells plated into a T225 flask(sub-confluent) are washed once with PBS and the DNA mixture is added toeach flask. The cells are allowed to incubate for 30 minutes at 37° C.,5% CO₂. Following the incubation, 36 ml of complete DMEM with 80 μMchloroquine is added to each flask and allowed to incubate an additional3 hours. The medium is then aspirated and 24 ml of complete mediumcontaining 10% DMSO for exactly 2 minutes and then aspirated. The cellsare then washed 2 times with PBS and 30 ml of complete DMEM added toeach flask. The cells are then allowed to incubate over night. The nextday the cells are harvested by trypsinization and reseeded as neededdepending upon the type of assay to be performed.

[0246] A typical protocol for liposomal-mediated transfection as appliedto CHO cells is described as follows; Cells to be used for transfectionare split 24 hours prior to the transfection to provide flasks which are70-80% confluent at the time of transfection. A total of 10 μg of DNAwhich may include varying ratios of receptor DNA plus any additional DNAneeded (e.g. Go protein expression vector, reporter construct,antibiotic resistance marker, mock vector, etc.) is used to transfecteach 75 cm² flask of cells. Liposomal mediated transfection is carriedout according to the manufacturer's recommendations (LipofectAMINE,GibcoBRL, Bethesda, Md.). Transfected cells are harvested 24 h posttransfection and used or reseeded according the requirements of theassay to be employed.

[0247] A typical protocol for the electroporation method as applied toCos-7 cells is described as follows; Cells to be used for transfectionare split 24 hours prior to the transfection to provide flasks which aresubconfluent at the time of transfection. The cells are harvested bytrypsinization resuspended in their growth media and counted. 4×10

cells are suspended in 300 μl of DMEM and placed into an electroporationcuvette. 8 μg of receptor DNA plus 8 μg of any additional DNA needed(e.g. G_(o) protein expression vector, reporter construct, antibioticresistance marker, mock vector, etc.) is added to the cell suspension,the cuvette is placed into a BioRad Gene Pulser and subjected to anelectrical pulse (Gene Pulser settings: 0.25 kV voltage, 950 μFcapacitance). Following the pulse, 800 μl of complete DMEM is added toeach cuvette and the suspension transferred to a sterile tube. Completemedium is added to each tube to bring the final cell concentration to1×10⁵ cells/100 μl. The cells are then plated as needed depending uponthe type of assay to be performed.

[0248] A typical protocol for viral mediated expression of heterolgousproteins is described as follows for baculovirus infection of insect Sf9cells. The coding region of DNA encoding the receptor disclosed hereinmay be subcloned into pBlueBacIII into existing restriction sites orsites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined in by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C. The supernatant of theco-transfection plate may be collected by centrifugation and therecombinant virus plaque purified. The procedure to infect cells withvirus, to prepare stocks of virus and to titer the virus stocks are asdescribed in Pharminger's manual. Similar principals would in generalapply to mammalian cell expression via retro-viruses, Simliki forestvirus and double stranded DNA viruses such as adeno-, herpes-, andvacinia-viruses, and the like.

[0249] Microinjection of cRNA encoding for proteins of interest isuseful for the study of protein function in xenopus oocytes as well ascultured mammalian cells. A typical protocol for the preparation of cRNAand injection into xenopus oocytes can be found in the followingelectrophysiology section.

[0250] Stable Expression

[0251] Heterologous DNA can be stably incorporated into host cells,causing the cell to perpetually express a foreign protein. Methods forthe delivery of the DNA into the cell are similar to those describedabove for transient expression but require the co-transfection of anancillary gene to confer drug resistance on the targeted host cell. Theensuing drug resistance can be exploited to select and maintain cellsthat have taken up the heterologous DNA. An assortment of resistancegenes are available including but not restricted to Neomycin, Kanamycin,and Hygromycin. For the purposes of receptor studies, stable expressionof a heterologous receptor protein is carried out in, but notnecessarily restricted to, mammalian cells including, CHO, HEK293,LM(tk-), etc.

[0252] Cell Membrane Preparation

[0253] For binding assays, pellets of transfected cells are suspended inice-cold buffer (20 mM Tris.HCl, 5 mM EDTA, pH 7.4) and homogenized bysonication for 7 sec. The cell lysates are centrifuged at 200× g for 5min at 4° C. The supernatants are then centrifuged at 40,000× g for 20min at 4° C. The resulting pellets are washed once in the homogenizationbuffer and suspended in binding buffer (see methods for radioligandbinding). Protein concentrations are determined by the method ofBradford (1976) using bovine serum albumin as the standard. Bindingassays are usually performed immediately, however it is possible toprepare membranes in batch and store frozen in liquid nitrogen forfuture use.

[0254] Radioligand Binding Assays

[0255] Cells may be screened for the presence of endogenous humanreceptor by radioligand binding (described in detail below). Cells witheither no or a low level of the endogenous human receptor disclosedherein may be transfected with the exogenous receptor.

[0256] MCH1 binding experiments with membranes (20-40 μg membraneprotein) from transfected cells are performed with 0.1 nM [

I]Phe¹³-T¹⁹-MCH (Custom labeled by NEN) using incubation bufferconsisting of 50 mM Tris pH 7.4, 10 mM MgCl₂, 2 μg/ml aprotonin, 0.5 mMPMSF and 50 μg/ml bacitracin. Binding is performed at 25° C. for 1 hr.Incubations are terminated by rapid vacuum filtration over GF/C glassfiber filters, presoaked in 5% PEI using 50 mM Tris pH 7.4 containing0.01% triton X-100 as wash buffer. In all experiments nonspecificbinding is defined using 10 μM unlabeled MCH.

[0257] Functional Assays

[0258] Cells may be screened for the presence of endogenous mammalianreceptor using functional assays (described in detail below). Cells withno or a low level of endogenous receptor present may be transfected withthe exogenous receptor for use in the following functional assays.

[0259] A wide spectrum of assays can be employed to screen for receptoractivation. These range from traditional measurements of phosphatidylinositol, cAMP, Ca⁺⁺, and K⁺, for example; to systems measuring thesesame second messengers but which have been modified or adapted to behigher throughput, more generic, and more sensitive; to cell basedplatforms reporting more general cellular events resulting from receptoractivation such as metabolic changes, differentiation, and celldivision/proliferation, for example; to high level organism assays whichmonitor complex physiological or behavioral changes thought to beinvolved with receptor activation including cardiovascular, analgesic,orexigenic, anxiolytic, and sedation effects, for example.

[0260] Cyclic AMP (cAMP) Assay

[0261] The receptor-mediated stimulation or inhibition of cyclic AMP(cAMP) formation may be assayed in cells expressing the mammalianreceptors. Cells are plated in 96-well plates and incubated inDulbecco's phosphate buffered saline (PBS) supplemented with 10 mMHEPES, 1 mM isobutylmethylxanthine for 20 min at 37° C., in 5% CO₂. Testcompounds are added with or without 10 μM forskolin and incubated for anadditional 10 min at 37° C. The medium is then aspirated and thereaction stopped by the addition of 100 mM HCl. The plates are stored at4° C. for 15 min, and the cAMP content in the stopping solution measuredby radioimmunoassay. Radioactivity may be quantified using a gammacounter equipped with data reduction software.

[0262] Arachidonic Acid Release Assay

[0263] Cells expressing the mammalian receptor are seeded into 96 wellplates and grown for 3 days in HAM's F-12 with supplements.[³H]-arachidonic acid (specific activity=0.75 μCi/ml) is delivered as a100 μL aliquot to each well and samples were incubated at 37° C., 5% CO₂for 18 hours. The labeled cells are washed three times with 200 μL HAM'sF-12. The wells are then filled with medium (200 μL) and the assay isinitiated with the addition of peptides or buffer (22 μL). Cells areincubated for 30 min at 37° C., 5% CO₂. Supernatants are transferred toa microtiter plate and evaporated to dryness at 75° C. in a vacuum oven.Samples are then dissolved and resuspended in 25 μL distilled water.Scintillant (300 μL) is added to each well and samples are counted for³H in a Trilux plate reader. Data are analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

[0264] Intracellular Calcium Mobilization Assay

[0265] The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Cells are seeded onto a 35 mm culture dishcontaining a glass coverslip insert, washed with HBS and loaded with 100μL of Fura-2/AM (10 μM) for 20 to 40 min. After washing with HBS toremove the Fura-2/AM solution, cells are equilibrated in HBS for 10 to20 min. Cells are then visualized under the 40× objective of a LeitzFluovert FS microscope and fluorescence emission is determined at 510 nMwith excitation wavelengths alternating between 340 nM and 380 nM. Rawfluorescence data are converted to calcium concentrations using standardcalcium concentration curves and software analysis techniques.

[0266] Inositol Phosphate Assay

[0267] Guidelines for cell preparation and assay of the second messengerinositol phosphate (IP) are described below for a typical protocolinvolving transiently transfected Cos-7 cells; For a 96 well microplateformat assay, cells are plated at 70,000 cells per well and allowed toincubate for 24 hours after the transfection procedure. The cells arethen labeled with 0.5 μCi [³H]myo-inositol per micro-well over night at37° C., 5% CO₂. Immediately before the assay, the medium is removed andreplaced with 90 μl PBS containing 10 mM LiCl. The plates are thenincubated for 15 minutes at 37° C., 5% CO₂. Following the incubation,the transfectants are challenged with agonist (10 μl/well; 10×concentration) for 30 minutes at 37° C., 5% CO₂. The challenge isterminated and the cells lysed by the addition of 100 μl cold 5% v/vtrichloroacetic acid (TCA), followed by an incubation at 4° C. forgreater than 30 minutes. Total IPs are isolated from the lysate by ionexchange chromatography. Briefly, the lysed contents of the wells aretransferred to a Multiscreen HV filter plate (Millipore) containing 100μl Dowex-AG1-X8 suspension (50% v/v, water:resin) (200-400 mesh, formateform). The filter plates are placed on a vacuum manifold to wash andelute the resin bed. Each well is first washed 2 times with 200 μl 5 mMmyoinositol. Total [³H]IPs are eluted with 75 μl of 1.2 M ammoniumformate/0.1 M formic acid into Wallac 96-well plates. 200 μl of SuperMixscintillation cocktail is added to each well, mixed well, allowed toequilibrate and counted on a Micro Beta Trilux scintillation counter.(Note: The assay may be scaled to a 24 well format by simple adjustmentof reagent volumes and employing individual chromatographic columns.)

[0268] GTPγS Functional Assay

[0269] Membranes from cells transfected with the mammalian receptors aresuspended in assay buffer (50 MM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.4)supplemented with 0.2% BSA and 10 μM GDP. Membranes are incubated on icefor 20 minutes, transferred to a 96-well Millipore microtiter GF/Cfilter plate and mixed with GTPγ³⁵S (e.g., 250,000 cpm/sample, specificactivity ˜1000 Ci/mmol) plus or minus GTPγS (final concentration=100μM). Final membrane protein concentration=90 μg/ml. Samples areincubated in the presence or absence of MCH (final concentration=1 μM)for 30 min. at room temperature, then filtered on a Millipore vacuummanifold and washed three times with cold assay buffer. Samplescollected in the filter plate are treated with scintillant and countedfor ³⁵S in a Trilux (Wallac) liquid scintillation counter. It isexpected that optimal results are obtained when the mammalian receptormembrane preparation is derived from an appropriately engineeredheterologous expression system, i.e., an expression system resulting inhigh levels of expression of the mammalian receptor and/or expressingG-proteins having high turnover rates (for the exchange of GDP for GTP).GTPγS assays are well-known in the art, and it is expected thatvariations on the method described above, such as are described by e.g.,Tian et al. (1994) or Lazareno and Birdsall (1993), may be used by oneof ordinary skill in the art.

[0270] Transcription Assay

[0271] Guidelines for cell preparation and assay of receptor mediatedtranscription of Cos-7 cells transiently transfected by the DEAE-dextranmethod in a 96 microwell format is as follows; The c-fos-β-galpromoter/reporter construct used for these studies consists of the cfospromoter region (−384 to +19) (Schilling et al 1991, Yalkinoglu et al,1995) inserted upstream of β-galactosidase cDNA containing expressionvector pNASSβ (Clontech). Transcription activity is measured by assay ofβ-galactosidase enzyme activity as detected in a colorimetric assay.Forty-eight hours following transient transfection, the medium isremoved and replaced with medium containing drug (e.g. MCH) typically ata concentration of 10 μM. The cells are allowed to incubate at 37° C.,5% CO₂ for at least 18 hours, after which the medium is aspirated andthe cells washed with 200 μl PBS/well. The cells are then lysed with 100μl AB buffer (100 mM Sodium Phosphate buffer, pH 8.0, 2 mM MgSO₄, 0.1 mMMnCl₂) for 10 minutes at room temperature. 100 μl ofAB/Tx/β-mercaptoethanol (AB buffer with 0.5% Triton X-100, 40 mMβ-mercaptoethanol) is then added to each well and the lysate allowed toincubate an additional 10 minutes at room temperature. The enzymaticcolor reaction is initiated by the addition of the substrate, ONPG/AB (4mg/ml O-nitrophenyl-b-D-galactopyranoside in AB buffer). The reaction isallowed to proceed for 30 minutes or until yellow color becomes evident.Measurement of optical density is taken at 405 nm using a Dynatechmicroplate reader.

[0272] MAP Kinase Assay

[0273] MAP kinase (mitogen activated kinase) may be monitored toevaluate receptor activation. MAP kinase is activated by multiplepathways in the cell. A primary mode of activation involves theras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase)receptors feed into this pathway via SHC/Grb-2/SOS/ras. Gi coupledreceptors are also known to activate ras and subsequently produce anactivation of MAP kinase. Receptors that activate phospholipase C (Gqand G11) produce diacylglycerol (DAG) as a consequence of phosphatidylinositol hydrolysis. DAG activates protein kinase C which in turnphosphorylates MAP kinase.

[0274] MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the mitogen and then extracted with Laemmli buffer. Thesoluble fraction is applied to an SDS-PAGE gel and proteins aretransferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

[0275] Another approach is based on evaluation of the MAP kinaseactivity via a phosphorylation assay. Cells are stimulated with themitogen and a soluble extract is prepared. The extract is incubated at30° C. for 10 min with gamma-32-ATP, an ATP regenerating system, and aspecific substrate for MAP kinase such as phosphorylated heat and acidstable protein regulated by insulin, or PHAS-I. The reaction isterminated by the addition of H₃PO₄ and samples are transferred to ice.An aliquot is spotted onto Whatman P81 chromatography paper, whichretains the phosphorylated protein. The chromatography paper is washedand counted for ³²P pin a liquid scintillation counter. Alternatively,the cell extract is incubated with gamma-32-ATP, an ATP regeneratingsystem, and biotinylated myelin basic protein bound by streptavidin to afilter support. The myelin basic protein is a substrate for activatedMAP kinase. The phosphorylation reaction is carried out for 10 min at30° C. The extract can then be aspirated through the filter, whichretains the phosphorylated myelin basic protein. The filter is washedand counted for ³²P by liquid scintillation counting.

[0276] Cell Proliferation Assay

[0277] Activation of a G protein coupled receptor may lead to amitogenic or proliferative response which can be monitored via[³H]-thymidine uptake. When cultured cells are incubated with[³H]-thymidine, the thymidine translocates into the nuclei where it isphosphorylated to thymidine triphosphate. The nucleotide triphosphate isthen incorporated into the cellular DNA at a rate that is proportionalto the rate of cell growth. Typically, cells are grown in culture for1-3 days. Cells are forced into quiescence by the removal of serum for24 hrs. A mitogenic agent is then added to the media. 24 hrs later, thecells are incubated with [³H]-thymidine at specific activities rangingfrom 1 to 10 μCi/ml for 2-6 hrs. Harvesting procedures may involvetrypsinization and trapping of cells by filtration over GF/C filterswith or without a prior incubation in TCA to extract soluble thymidine.The filters are processed with scintillant and counted for ³H by liquidscintillation counting. Alternatively, adherent cells are fixed in MeOHor TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 NNaOH. The soluble extract is transferred to scintillation vials andcounted for ³H by liquid scintillation counting.

[0278] Methods for Recording Currents in Xenopus Oocytes

[0279] Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) areanesthetized in 0.2% tricain (3-aminobenzoic acid ethyl ester, SigmaChemical Corp.) and a portion of ovary is removed using aseptictechnique (Quick and Lester, 1994) Oocytes are defolliculated using 2mg/ml collagenase (Worthington Biochemical Corp., Freehold, N.J.) in asolution containing 87.5 mM NaCl, 2 mM KCl, 2 mM MgCl and 5 mM HEPES, pH7.5. Oocytes may be injected (Nanoject, Drummond Scientific, Broomall,Pa.) with mammalian mRNA. Other oocytes may be injected with a mixtureof mammalian mRNA and mRNA encoding the genes for G-protein-activatedinward rectifiers (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and5,728,535). Genes encoding G-protein inwardly rectifying K⁺ (GIRK)channels 1 and 4 (GIRK1 and GIRK4) were obtained by PCR using thepublished sequences (Kubo et al., 1993; Dascal et al., 1993; Krapivinskyet al., 1995 and 1995b) to derive appropriate 5′ and 3′ primers. Humanheart cDNA was used as template together with the primers5′-CGCGGATCCATTATGTCTGCACTCCGAAGGA (SEQ ID NO: 10) AATTTG-3′ and5′-CGCGAATTCTTATGTGAAGCGATCAGAGTTC (SEQ ID NO: 11) ATTTTTC- 3′ for GIRK1and 5′-GCGGGATCCGCTATGGCTGGTGATTCTAGGA (SEQ ID NO: 12) ATG- 3′ and5′-CCGGAATTCCCCTCACACCGAGCCCCT (SEQ ID NO: 13) GG-3′ for GIRK4.

[0280] In each primer pair, the upstream primer contained a BamHI siteand the downstream primer contained an EcoRI site to facilitate cloningof the PCR product into pcDNA1-Amp (Invitrogen). The transcriptiontemplate for the mammalian receptor may be similarly obtained. mRNAs areprepared from separate DNA plasmids containing the complete codingregions of the mammalian receptor, GIRK1, and GIRK4. Plasmids arelinearized and transcribed using the T7 polymerase (“Message Machine”,Ambion). Alternatively, mRNA may be translated from a template generatedby PCR, incorporating a T7 promoter and a poly A⁺ tail. Each oocytereceives 2 ng each of GIRK1 and GIRK4 mRNA in combination with 25 ng ofmammalian receptor mRNA. After injection of mRNA, oocytes are incubatedat 16° C. on a rotating platform for 3-8 days. Dual electrode voltageclamp (“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) isperformed using 3 M KCl-filled glass microelectrodes having resistancesof 1-3 Mohms. Unless otherwise specified, oocytes are voltage clamped ata holding potential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 2 mM MgCl

, and 5 mM HEPES, pH 7.5 (“ND96”), or, in the case of oocytes expressingGIRK1 and GIRK4, elevated K⁺ containing 96 mM KCl, 2 mM NaCl, 2 mMCaCl₂, 2 mM MgCl

, and 5 mM HEPES, pH 7.5 (“hK”). Drugs are applied by switching from aseries of gravity fed perfusion lines.

[0281] Heterologous expression of GPCRs in Xenopus oocytes has beenwidely used to determine the identity of signaling pathways activated byagonist stimulation (Gundersen et al., 1983; Takahashi et al., 1987).Activation of the phospholipase C (PLC) pathway is assayed by applyingtest compound in ND96 solution to oocytes previously injected with mRNAfor the mammalian receptor and observing inward currents at a holdingpotential of −80 mV. The appearance of currents that reverse at −25 mVand display other properties of the Ca⁻⁻-activated Cl⁻ (chloride)channel is indicative of mammalian receptor-activation of PLC andrelease of IP3 and intracellular Ca⁺⁺. Such activity is exhibited byGPCRs that couple to G_(q).

[0282] Measurement of inwardly rectifying K⁺ (potassium) channel (GIRK)activity is monitored in oocytes that have been co-injected with mRNAsencoding the mammalian receptor, GIRK1, and GIRK4. The two GIRK geneproducts co-assemble to form a G-protein activated potassium channelknown to be activated (i.e., stimulated) by a number of GPCRs thatcouple to G, or GQ (Kubo et al., 1993; Dascal et al., 1993). Oocytesexpressing the mammalian receptor plus the two GIRK subunits are testedfor test compound responsivity by measuring K⁺ currents in elevated K⁺solution (hK). Activation of inwardly rectifying currents that aresensitive to 300 μM Ba signifies the mammalian receptor coupling to a G

or G

pathway in the oocytes.

[0283] Receptor/G Protein Co-Transfection Studies

[0284] A strategy for determining whether MCH1 can couple preferentiallyto selected G proteins involves co-transfection of MCH1 receptor cDNAinto a host cell together with the cDNA for a G protein alpha sub-unit.Examples of G alpha sub-units include members of the Gαi/Gαo class(including Gαt2 and Gαz), the Gαq class, the Gαs class, and the Gα12/13class. A typical procedure involves transient transfection into a hostcell such as COS-7. Other host cells may be used. A key consideration iswhether the cell has a downstream effector (a particular adenylatecyclase, phospholipase C, or channel isoform, for example) to support afunctional response through the G protein under investigation. G proteinbeta gamma sub-units native to the cell are presumed to complete the Gprotein heterotrimer; otherwise specific beta and gamma sub-units may beco-transfected as well. Additionally, any individual or combination ofalpha, beta, or gamma subunits may be co-transfected to optimize thefunctional signal mediated by the receptor.

[0285] The receptor/G alpha co-transfected cells are evaluated in abinding assay, in which case the radioligand binding may be enhanced bythe presence of the optimal G protein coupling or in a functional assaydesigned to test the. receptor/G protein hypothesis. In one example, theMCH1 receptor may be hypothesized to inhibit cAMP accumulation throughcoupling with G alpha sub-units of the Gαi/Gαo class. Host cellsco-transfected with the MCH1 receptor and appropriate G alpha sub-unitcDNA are stimulated with forskolin ±MCH1 agonist, as described above incAMP methods. Intracellular cAMP is extracted for analysis byradioimmunoassay. Other assays may be substituted for cAMP inhibition,including GTPγ³⁵S binding assays and inositol phosphate hydrolysisassays. Host cells transfected with MCH1 minus G alpha or with G alphaminus MCH1 would be tested simultaneously as negative controls. MCH1receptor expression in transfected cells may be confirmed in radioligandbinding studies using membranes from transfected cells. G alphaexpression in transfected cells may be confirmed by Western blotanalysis of membranes from transfected cells, using antibodies specificfor the G protein of interest.

[0286] The efficiency of the transient transfection procedure is acritical factor for signal to noise in an inhibitory assay, much more sothan in a stimulatory assay. If a positive signal present in all cells(such as forskolin-stimulated cAMP accumulation) is inhibited only inthe fraction of cells successfully transfected with receptor and Galpha, the signal to noise ratio will be poor. One method for improvingthe signal to noise ratio is to create a stably transfected cell line inwhich 100% of the cells express both the receptor and the G alphasubunit. Another method involves transient co-transfection with a thirdcDNA for a G protein-coupled receptor which positively regulates thesignal which is to be inhibited. If the co-transfected cellssimultaneously express the stimulatory receptor, the inhibitoryreceptor, and a requisite G protein for the inhibitory receptor, then apositive signal may be elevated selectively in transfected cells using areceptor-specific agonist. An example involves co-transfection of COS-7cells with 5-HT4 receptor, MCH1 receptor, and a G alpha sub-unit.Transfected cells are stimulated with a 5-HT4 agonist ±MCH1 agonist.Cyclic AMP is expected to be elevated only in the cells also expressingMCH1 and the G alpha subunit of interest, and a MCH1-dependentinhibition may be measured with an improved signal to noise ratio.

[0287] It is to be understood that the cell lines described herein aremerely illustrative of the methods used to evaluate the binding andfunction of the mammalian receptors of the present invention, and thatother suitable cells may be used in the assays described herein.

[0288] Promiscuous Second Messenger Assays

[0289] It is possible to coax receptors of different functional classesto signal through a pre-selected pathway through the use of promiscuousG_(o) subunits. For example, by providing a cell based receptor assaysystem with an exogenously supplied promiscuous G_(o) subunit such asG_(o) or a chimeric G

subunit such as G_(ozq), a GPCR which normally might prefer to couplethrough a specific signaling pathway (e.g. G_(s), G

, G_(q), G

etc.), can be made to couple through the pathway defined by thepromiscuous G_(o) subunit and upon agonist activation produce the secondmessenger associated with that subunit's pathway. In the case of G_(o16)and/or G

this would involve activation of the G_(q) pathway and production of thesecond messenger inositol phosphate. Through similar strategies andtools, it is possible to bias receptor signaling through pathwaysproducing other second messengers such as Ca⁺⁺, cAMP, K⁺ currents, etc.

[0290] Microphysiometric Assay

[0291] Because cellular metabolism is intricately involved in andeffected by a broad range of cellular events (including receptoractivation of various, second messenger pathways), the use ofmicrophysiometric measurements of cell metabolism can in principleprovide a generic assay of cellular activity arising from the activationof any receptor regardless of the specifics of the receptor's proximalsignaling pathway.

[0292] General guidelines for cell preparation and microphysiometricrecording have been previously reported (Salon, J. A. and Owicki, J. A.,1996). A typical protocol employing transiently transfected CHO cells isas follows; 24 hours prior to recording, transfected cells are harvestedand counted. 3×10⁵ cells are seeded into cell culture capsules (Costar),and allowed to attach to the capsule membrane. 10 hours later (14 hoursprior to recording) the cell media is switched to serum free F-12complete to minimize ill-defined metabolic stimulation caused byassorted serum factors.

[0293] On the day of the experiment the cell capsules are transferred tothe microphysiometer (Cytosensor, Molecular Devices Corporation,Sunnyvale, Calif.) and allowed to equilibrate in recording media (lowbuffered RPMI 1640, no bicarbonate, no serum) with 0.1% BSA (essentiallyfatty acid free), during which a baseline measurement of basal metabolicactivity is established. The recording paradigm consists of a 100 μl/minflow rate, with a 2 min pump cycle which includes a 30 sec flowinterruption during which the rate measurement is taken. Challengesinvolve a 1 min 20 sec exposure to a drug just prior to the first postchallenge rate measurement being taken, followed by two additional pumpcycles for a total of 5 min 20 sec drug exposure. Drug is then washedout and rates allowed to return to basal. Reported extracellularacidification rates are expressed as a percentage increase of the peakresponse over the baseline rate observed just prior to challenge.

[0294] GPCR Licand Library

[0295] Functional assays of new receptors such as MCH1 may include apreliminary test of a small library of compounds containingrepresentative agonists for all known GPCRs as well as other compoundswhich may be agonists for prospective GPCRs or which may be effectorsfor targets peripherally involved with GPCRs. The collection used inthis study comprises approximately 180 compounds (including smallmolecules, hormones, preprohormones, peptides, etc.) for more than 45described classes of GPCRs (serotonin, dopamine, noradrenaline, opioids,etc.) and additionally includes ligands for known or suspected but notnecessarily pharmacological characterized or cloned GPCR families (suchas MCH).

[0296] The diversity of the library can be expanded to include agonistand antagonist compounds specific for GPCR subtypes, combinatorialpeptide and/or small molecule libraries, natural product collections,and the like. To facilitate robotic handling, the substances aredistributed as either separate or pooled compound concentrates in 96well plates and stored frozen as ready to use reagent plates.

[0297] Localization of mRNA Coding for Human MCH1 Receptors

[0298] Development of probes for MCH1: To facilitate the production ofradiolabeled, antisense RNA probes a fragment of the gene encoding ratMCH1 will be subcloned into a plasmid vector containing RNA polymerasepromoter sites. The full length cDNA encoding the rat MCH1 will bedigested with Pst 1, (nucleotides 905-1194) and this 289 nucleotidefragment will be cloned into the Pst I site of pGEM 3z, containing bothsp6 and T7 RNA polymerase promoter sites. The construct will besequenced to confirm sequence identity and orientation. To synthesizeantisense strands of RNA, this construct will be linearized with HindIII or Eco RI (depending on orientation) and T7 or sp6 RNA polymerasewill be used to incorporate radiolabeled nucleotide as described below.

[0299] A probe coding for the rat glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene, a constitutively expressed protein, was usedconcurrently. GAPDH is expressed at a relatively constant level in mosttissue and its detection is used to compare expression levels of the ratMCH1 receptors gene in different regions.

[0300] Synthesis of probes: MCH1 and GAPDH cDNA sequences preceded byphage polymerase promoter sequences will be used to synthesizeradiolabeled riboprobes. Conditions for the synthesis of riboprobes willbe: 0.25-1.0 μg linearized DNA plasmid template, 1.5 μl of ATP, GTP, UTP(10 mM each), 3 μl dithiothreitol (0.1 M), 30 units RNAsin RNAseinhibitor, 0.5-1.0 μl (15-20 units/μl) RNA polymerase, 7.0 μltranscription buffer (Promega Corp.), and 12.5 μl α P-CTP (specificactivity 3,000 Ci/mmol). 0.1 mM CTP (0.02-1.0 μl) will be added to thereactions, and the volume will be adjusted to 35 μl with DEPC-treatedwater. Labeling reactions will be incubated at 37° C. for 60 min, afterwhich 3 units of RQ1 RNAse-free DNAse (Promega Corp.) will be added todigest the template. Riboprobes will be separated from unincorporatednucleotides using Microspin S-300 columns (Pharmacia Biotech). TCAprecipitation and liquid scintillation spectrometry will be used tomeasure the amount of label incorporated into the probe. A fraction ofall riboprobes synthesized will be size-fractionated on 0.25 mm thick 7Murea, 4.5% acrylamide sequencing gels. These gels will be apposed tostorage phosphor screens and the resulting autoradiograph scanned usinga phoshorimager (Molecular Dynamics, Sunnyvale, Calif.) to confirm thatthe probes synthesized were full-length and not degraded.

[0301] Solution hybridization/ribonuclease protection assay (RPA): Forsolution hybridization 2.0 μg of mRNA isolated from tissues will beused. Negative controls consisted of 30 μg transfer RNA (tRNA) or notissue blanks. All mRNA samples will be placed in 1.5-ml microfuge tubesand vacuum dried. Hybridization buffer (40 μl of 400 mM NaCl, 20 mMTris, pH 6.4, 2 mM EDTA, in 80% formamide) containing 0.25-2.0 E⁶ countsof each probe will be added to each tube. Samples will be heated at 95°C. for 15 min, after which the temperature will be lowered to 55° C. forhybridization.

[0302] After hybridization for 14-18 hr, the RNA/probe mixtures will bedigested with RNAse A (Sigma) and RNAse T1 (Life Technologies). Amixture of 2.0 μg RNAse A and 1000 units of RNAse T1 in a buffercontaining 330 mM NaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) willbe added to each sample and incubated for 90 min at room temperature.After digestion with RNAses, 20 μl of 10% SDS and 50 μg proteinase Kwill be added to each tube and incubated at 37° C. for 15 min. Sampleswill be extracted with phenol/chloroform:isoamyl alcohol andprecipitated in 2 volumes of ethanol for 1 hr at −70° C. Pellet Paint(Novagen) will be added to each tube (2.0 μg) as a carrier to facilitateprecipitation. Following precipitation, samples will be centrifuged,washed with cold 70% ethanol, and vacuum dried. Samples will bedissolved in formamide loading buffer and size-fractionated on aurea/acrylamide sequencing gel (7.0 M urea, 4.5% acrylamide inTris-borate-EDTA). Gels will be dried and apposed to storage phosphorscreens and scanned using a phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

[0303] RT-PCR: For the detection of RNA encoding human MCH1, RT-PCR wascarried out on mRNA extracted from human tissue. Reverse transcriptionand PCR reactions were carried out in 50 ml volumes using EZrTth DNApolymerase (Perkin Elmer). Primers with the following sequences wereused: Forward primer (RA SLC1a /MCH F); TCA GCT CGG TTG TGG GAG CA (SEQID NO: 14) Reverse primer (RA/SLC1a MCH B); CTT GGA CTT CTT CAC GAC (SEQID NO: 15)

[0304] These primers will amplify a 248 base pair fragment fromnucleotide 169 to 417.

[0305] Each reaction contained 0.1 μg mRNA and 0.3μM of each primer.Concentrations of reagents in each reaction were: 300 μM each of GTP;DATP; dCTP; dTTP; 2.5 mM Mn(OAc)2; 50 mM Bicine; 115 mM potassiumacetate, 8% glycerol and 5 units EZrTth DNA polymerase. All reagents forPCR (except mRNA and oligonucleotide primers) were obtained from PerkinElmer. Reactions were carried out under the following conditions: 65° C.60 min., 94° C. 2 min., (94° C., 1 min., 65° C. 1 min) 35 cycles, 72° C.10 min. PCR reactions were size fractionated by gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene Oreg.) and scanned on a Molecular Dynamics (SunnyvaleCalif.) Storm 860 in blue fluorescence mode at 450 nM.

[0306] Positive controls for PCR reactions consisted of amplification ofthe target sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks, as well as primer and mRNA blanks. To confirmthat the mRNA was not contaminated with genomic DNA, samples weredigested with RNAses before reverse transcription. Integrity of RNA wasassessed by amplification of mRNA coding for GAPDH.

[0307] Results and Discussion

[0308] Cloning and Sequencing

[0309] Discovery of an Expressed Sequence Tac (EST) F07228 in GENEMLHomologous to FB41a

[0310] A BLAST search of GENEMBL with a Synaptic Pharmaceuticalcorporation proprietary sequence, FB41a, resulted in the identificationof an EST (accession number F07228) with a high degree of homology toFB41a and somatostatin, opiate and galanin receptors.

[0311] Construction and Screening of a Human Hippocampal cDNA Library

[0312] A human hippocampal cDNA library containing a total of 2.2 x10independent clones with a mean insert size of 3.0 kb was prepared in theexpression vector pEXJ.BS. The library was plated on agar plates(ampicillin selection) and glycerol stocks for 450 pools of 5000independent clones were prepared. Primary glycerol stocks were alsogrouped together in groups of approximately 10 to create superpools.

[0313] Cloning of the Full-Length Sequence of MCH1

[0314] Glycerol stocks of the superpools and primary pools from thehuman hippocampal cDNA library were screened by PCR with F07228 specificprimers T579 and T580. One positive primary pool 490, was successivelydivided into subpools, amplified in LB medium overnight and screened byPCR using primers T579 and T580. One positive subpool, 490-4-10-23 wasplated on agar plates (ampicillin selection), and colonies weretransferred to nitrocellulose membranes (Schleicher and Schuell, Keene,N.H.). Filters were hybridized for two days under high stringencyconditions with 10⁶ cpm/ml of a ³²P-labeled cDNA probe, T581, designedagainst the F07228 EST sequence. Filters were washed and apposed toBiomax MS film (Kodak). Seven positive colonies were picked, streaked onLB-AMP plates, and grown overnight. Two individual colonies from each ofthe original seven were picked and subjected to vector-anchored PCRusing the following primer pairs: T95, T580 and T94, T579. One positivecolony, G1, was amplified overnight in TB and processed for plasmidpurification. This plasmid was designated TL230 and sequenced on bothstrands. Nucleotide and peptide sequence analysis were performed withGCG programs (Genetics Computer Group, Madison, WI). A HindIII-KpnIfragment of TL230 was subcloned into the mammalian expression vectorpEXJ, and named TL231. The largest open reading frame in this constructcontains 1266 nucleotides (FIG. 1), which is predicted to encode aprotein of 422 amino acids (FIG. 2). There are three in-framemethionines in the amino terminus which could result in a protein of422, 417 or 353 amino acids. Hydropathy analysis of the protein isconsistent with a putative topography of seven transmembrane domains,indicative of the G protein-coupled receptor family (FIG. 3). TL231 hasbeen named MCH1.

[0315] Database analysis of the sequence of MCH1 revealed that it wasmost similar to somatostatin receptors. Further database analysisrevealed a Genbank submission (accession number AF008650, deposited onOct. 1, 1997) which appears to be the rat homologue of TL231. AF008650is 69 nucleotides shorter than MCH1 at the 5′ end, and predicts adifferent initiating methionine. FIGS. 4 and 5 illustrate the nucleotideand amino acid sequence for the rat MCH1 receptor, respectively.

[0316] Inositol Phosphate Response of MCH1-Transfected Cells

[0317] The expression vector (pEXJ) containing the MCH1 cDNA wastransfected by electroporation into Cos-7 cells in combination with anexpression vector (pEXJ) containing the G

, subunit. After plating and labeling with [³H]-myo-inositol, thetransfectants were challenged with a ligand library that included, amongother things, melanin concentrating hormone (MCH) (10 μM finalconcentration) and then assayed for inositol phosphate (IP) formation.In five out of the seven screens, cells transfected with MCH1 (withG_(old)) gave an approximately 1.4-fold increase in IP production ascompared to cells transfected with G

alone when challenged with MCH.

[0318] Subsequent experiments demonstrated that 10 μM MCH was able tostimulate IP release 3.4-fold over basal levels in Cos-7 cellstransfected with MCH1 alone, suggesting that this receptor couplesthrough the G_(q) signaling pathway. The IP response was shown to bedose-dependent to MCH with an EC

value of 9.3±1.7 nM (n=2) and an E_(max) of approximately 400% basal(404±72) (FIG. 6).

[0319] Several additional compounds were tested for their ability toactivate MCH1. No dose-responsiveness of inositol phosphate formationcould be detected in Cos-7 cells transfected with MCH1 when challengedwith somatostatin, haloperidol, or dynorphin A1-13, discounting thepossibility that MCH1 encodes a somatostatin-like or opioid-like orsigma-like GPCR subtype (FIG. 7)

[0320] Microphysiometric Response of MCP1-Transfected Cells to MCH

[0321] CHO cells were transiently transfected with MCH1 usinglipofectant, challenged with increasing concentrations of MCH orPhe¹³,Tyr¹⁹-MCH, and subsequently monitored for changes in extracellularacidification rates. Both ligands produced a dose-dependent increase inacidification rate with an EC₅₀ value of 8.6 nM for MCH and 51.8 nM forPhe¹³,Tyr¹⁹-MCH. Neither native CHO cells or mock (PEXJ) transfected CHOcells exhibited a change in acidification rate when exposed to MCH orPhe¹³,Tyr¹⁹-MCH (FIG. 8)

[0322] Transcriptional Response of MCH1-Transfected Cells

[0323] Cos-7 cells were transiently transfected with MCH1 and ac-fos-β-gal reporter construct by the DEAE-dextran method. The cellswere challenged with assorted drugs, including MCH, and transcriptionalactivity measured by colorimetric assay of β-galactosidase proteinexpression. Initial single dose challenges with MCH at a concentrationof 10 μM stimulated c-fos-regulated transcriptional activityapproximately 3.9-fold over cells challenged with medium only. Cellstransfected with only the c-fos-β-gal construct showed no response toMCH. Subsequent experimentation showed the transcription activationresponse to be dose-dependent to MCH with an EC₅₀ value of 116 nM (FIG.9).

[0324] Binding of [¹²⁵I]Phe¹³,Tyr¹⁹-MCH in MCH1-Transfected Cells

[0325] Membranes harvested from Cos-7 cells transfected with MCH1 by theDEAE-dextran method exhibited specific binding for [¹²⁵I]Phe¹³-Tyr¹⁹-MCH(about 80 fmol/mg membrane protein) over mock-transfected cells (about20 fmol/mg membrane protein) at 0.1 nM radioligand concentration.Specific [¹²⁵I]Phe¹³-Tyr¹⁹-MCH binding was about 70% of total bindingat. a radioligand concentration of 0.1 nM (FIG. 10).

[0326] Localization of mRNA Encoding Human MCH1 Receptors

[0327] RT-PCR was used to assess the presence of MCH1 receptor encodingmessage in mRNA samples isolated from a variety of human tissues (Table1, FIG. 11). After amplification, PCR reactions were size fractionatedon 10% polyacrylamide gels, and stained with SYBR Green I. Images wereanalyzed using a Molecular Dynamics Storm 860 workstation. The amplifiedband corresponding to MCH1 receptor (490 base pairs) is indicated(arrow). RT-PCR analysis indicates the distribution of mRNA encodinghuman MCH1 receptor is widespread throughout all tissues assayed,including both central nervous system tissue and peripheral organs. Thiswidespread distribution implies broad regulatory functions that involvenervous system as well as endocrine mechanisms. TABLE 1 Distribution ofmRNA coding for human MCH1 receptors. human Region MCH 1 Potentialapplications liver +++ Diabetes kidney +++ Hypertension, Electrolytebalance lung +++ Respiratory disorders, asthma heart +++ Cardiovascularindications small intestine +++ Gastrointestinal disorders striatedmuscle +++ Musculoskeletal disorders pituitary +++Endocrine/neuroendocrine regulation whole brain +++ amygdala +++Depression, phobias, anxiety, mood disorders cerebral cortex +++ Sensoryand motor integration, cognition hippocampus +++ Cognition/memoryhypothalamus +++ appetite/obesity, neuroendocrine regulation spinal cord+++ Analgesia, sensory modulation and transmission cerebellum +++ Motorcoordination thalamus +++ sensory integration substantia +++ Modulationof dopaminergic nigra function. Modulation of motor coordination.caudate-putamen +++ Modulation of dopaminergic function fetal brain +++Developmental disorders fetal lung +++ Developmental disorders fetalkidney +++ Developmental disorders fetal liver +++ Developmentaldisorders

[0328] The cloning of the gene encoding the human MCH1 receptor hasprovided the means to explore its physiological role by pharmacologicalcharacterization, and by Northern and in situ mapping of its mRNAdistribution. Further, the availability of the DNA encoding the humanMCH1 receptor will facilitate the development of antibodies andantisense technologies useful in defining the functions of the geneproducts in vivo. Antisense oligonucleotides which target mRNA moleculesto selectively block translation of the gene products in vivo have beenused successfully to relate the expression of a single gene with itsfunctional sequelae. Thus, the cloning of this receptor gene providesthe means to explore its physiological role in the nervous system andelsewhere, and may thereby help to elucidate structure/functionrelationships within the GPCR superfamily.

[0329] he presence of three different potential starting codons in thecDNA sequence of TL231 opens the question of which of the possibletranscripts yields an active MCH receptor. In order to establish whethera transcript of the first and second starting codons of TL231 encode afunctional human MCH receptor, methionines 6 and 70 of TL231 weremutated to alanine (construct R114; See FIG. 12). The third methionineat position 70 was also mutated to an alanine (construct R106; See FIG.12). Transfections of TL231, R106 or R114 into COS-7 cells all resultedin MCH-mediated increases of intracellular calcium, as measured by afluorescent intensity plate reader in cells loaded with the calcium dyefluo-3 (FLIPR, Molecular Devices). As shown in Table 2, COS-7 cellstransfected with TL231, R106, R114 and BO120 showed dose-relatedmobilization of intracellular calcium when exposed to increasingconcentrations of MCH with similar maximal responses and EC50 values.These data demonstrate that transcripts starting at the first and/orsecond and third methionine of TL231 encode a functional human MCHreceptor. TABLE 2 Response to Melanin Concentrating Transfected Hormone*Construct EC50 (nM) Max. Response (RFU**) TL231 60, 12  3,535, 14,000R114 98, 9  2,267, 1,550 R106 85, 55   4642, 2000 BO120 12, 3.5 30,000,25,000

[0330] References

[0331] Abrao, M. S., Castrucci, A. M., Hacley, M. E. and Hruby, V. J.(1991) Protein-kinase-C mediates MCH signal transduction in teleost,Synbranchus marmoratus, melanocytes. Pigment. Cell. Res. 4:66-67.

[0332] Auburger, G., Gispert, S., Scheufler, K., Nothers, C., Lunkes,A., Hernandez, A., Magarino, C., Enczmann, J., Freund, H. J., Heredero,L., and Orozco, G. (1992) Assignment of the second (cuban) locus ofautosomal dominant cerebellar ataxia to chromosome 12q23-24.1, betweenflanking markers D12558 and PLA2. Cytogenet. Cell. Genet. 61:252-256.

[0333] Bahjaoui-Bouhaddi, M., Fellmann, D., Griffond, B. and Bugnon, C.(1994) Insulin treatment stimulates the rat melanin-concentratinghormone-producing neurons. Neuropeptides 24:251-258.

[0334] Baker, B. I. (1991) Melanin-concentrating hormone: a generalvertebrate neuropeptide. Int. Rev. Cytol. 126:1-47.

[0335] Baker, B. I. (1994) Melanin-concentrating hormone update:functional consideration. TEM 5:120-126.

[0336] Bassett, A. S., Jones, B. D., McGillivray, B. C. and Pantzer, J.T. (1988) Partial trisomy chromosome 5 cosegregating with schizophrenia.Lancet 1:799-801.

[0337] Bittencourt, J. C., Presse, F., Arias, C., Peto, C., Vaughan, J.,Nahon, J. L., Vale, W., Sawchenko, P. E. (1992) Themelanin-concentrating hormone system of the rat brain: An immuno- andhybridization histochemical characterization J. Comp. Neurol.319:218-245.

[0338] Bradford, M. M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal Biochem May 7, 1976; 72:248-54.

[0339] Breton, C., Schorpp, M., and Nahon, J. L. (1993) Isolation andcharacterization of the human melanin-concentrating hormone gene and avariant gene. Mol. Brain Res. 18:297-310.

[0340] Burgaud, J. L., Poosti, R., Fehrentz, J. A., Martinez, J., andNahon, J. L. (1997) Melanin-concentrating hormone binding sites in humanSVK14 keratinocytes. Biochem.Biophys.Res.Commun. 241(3) :622-629.

[0341] Burns, C. C., Moser, M., Banks, J., Alderete, J. P., andOverbaugh, J. (1996) Identification and deletion of sequences requiredfor feline leukemia virus RNA packaging and construction of a high-titerfeline leukemia virus packaging cell line. Virology (Aug. 1, 1996)222(1) :14-20.

[0342] Chu, Y. Y., Tu, K. H., Lee, Y. C., Kuo, Z. J., Lai, H. L., andChern, Y. (1996) Characterization of the rat A2a adenosine receptorgene. DNA Cell Biol (1996 Apr) 15(4) 329-37.

[0343] Coleman, A. (1984) Transcription and Translation: A PracticalApproach (B. D. Hanes, S. J. Higgins, eds., pp 271-302, IRL Press,Oxford, 1984).

[0344] Craddock, N., Dawson, E., Burge, S., Parfitt, L., Mant, B.,Roberts, Q., Daniels, J., Gill, M., McGuffin, P., Powell, J. and Owen,M. (1993) The gene for Darier's disease maps to chromosome 12q23-q24.1.Hum. Mol. Genet. 2:1941-1943.

[0345] Dascal, N., Schreibmayer, W., Lim, N. F., Wang, W., Chavkin, C.,DiMagno, L., Labarca, C., Kieffer, B. L., Gaveriaux-Ruff, C.,Trollinger, D., Lester, H. A., Davidson, N. (1993) Atrial Gprotein-activated K+ channel: expression cloning and molecularproperties. Proc. Natl. Acad. Sci. USA 90:10235-10239.

[0346] Drozdz, R. and Eberle, A. N. (1995) Binding sites formelanin-concentrating hormone (MCH) in brain synaptosomes and membranesfrom peripheral tissues identified with highly tritiated MCH.J.Recept.Signal.Transduct.Res. 15(1-4):487-502.

[0347] Drozdz, R., Siegrist, W., Baker, B. I., Chluba-de Tapia, J. andEberle, A. N. (1995) Melanin-concentrating hormone binding to mousemelanoma cells in vitro. FEBS 35:199-202.

[0348]

[0349] Drozdz, R., Hintermann, E., and Eberle, A. N. (1998)Characterization of the receptor for melanin-concentrating hormone onmelanoma cells by photocrosslinking. Ann.NY Acad.Sci. 83(1):210-213.

[0350] Fong, T. M.; Huang, R. C.; Yu, H.; Swain, C. J.; Underwood, D.;Cascieri, M. A.; Strader, C. D. (1995) Mutational analysis of neurokininreceptor function. Can. J. Physiol. Pharmacol. 73(7) :860-865 (Jul 1995)

[0351] Gilliam, T. C., Freimer, N. B., Kaufmann, C. A., Powchik, P. P.,Bassett, A. S., Bengtsson₁ U. and Wasmuth, J. J. (1989) Deletion mappingof DNA markers to a region of chromosome 5 that cosegregates withschizophrenia. Genomics 5:940-944.

[0352] Gonzalez, M. I., Baker, B. I., and Wilson, C. A. (1997)Stimulatory effect of melanin-concentrating hormone on luteinizinghormone release. Neuroendocrinology. 66(4):254-262.

[0353] Gonzalez, M. I., Kalia, V., Hole, D. R. and Wilson, C. A. (1997)α-melanocyte-stimulating hormone (α-MSH) and melanin-concentratinghormone (MCH) modify monoaminergic levels in the preoptic area of therat. Peptides 18:387-392.

[0354] Gonzalez, M. I., Vazira, S., and Wilson, C. A. (1996) Behavioraleffects of α-melanocyte-stimulating hormone (α-MSH) andmelanin-concentrating hormone (MCH) after central administration infemale rats. Peptides 17:171-177.

[0355] Graziano, M. P.; Hey, P. J.; Strader, C. D. (1996)The aminoterminal domain of the glucacon-like peptide-1 receptor is a criticaldeterminant of subtype specificity. Receptors Channels 4(1):9-17.

[0356] Grillon, S., Herve, C., Griffond, B., and Fellmann, D. (1997)Exploring the expression of the melanin-concentrating hormone messengerRNA in the rat lateral hypothalamus after goldthioglucose injection.Neuropeptides 31(2) :131-136.

[0357] Guan, X. M.; Amend, A.; Strader, C. D. (1995) Determination ofstructural domains for G protein coupling and ligand binding in beta3-adrenergic receptor. Mol. Pharmacol. 48(3) :492-498 (Sep 1995).

[0358] Gundersen, C. B., Miledi, R., and Parker, I. (1983) Serotoninreceptors induced by exogenous messenger RNA in Xenopus oocytes. Proc RSoc Lond B Biol Sci (Aug. 22, 1983) 219:1214 103-9.

[0359] Herve, C. and Fellmann, D. (1997) Changes in ratmelanin-concentrating hormone and dynorphin messenger ribonucleic acidsinduced by food deprivation. Neuropeptides 31(3):237-242.

[0360] Hervieu, G. and Nahon, J. L. (1995) Pro-melanin concentratinghormone messenger ribonucleic acid and peptides expression in peripheraltissues of the rat. Neuroendocrinology. 61(4):348-364.

[0361] Hervieu, G., Segretain, D. and Nahon, J-L. (i996) Development andstage-dependent expression of melanin-concentrating hormone in mammaliangerm cells. Biology of Reproduction 54:1161-1172.

[0362] Kauwachi, H., Kawazoe, I., Tsubokawa, M., Kishida, M. and Baker,B. I. (1983) Characterization of melanin-concentrating hormone in chumsalmon pituitaries. Nature 305:321-333.

[0363] Knigge, K. M., Baxter-Grillo, D., Speciale, J. and Wagner, J.(1996) Melanotropic peptides in the mammalian brain: Themelanin-concentrating hormone. Peptides 17:1063-1073.

[0364] Knigge, K. M. and Wagner, J. E. (1997) Melanin-concentratinghormone (MCH) involvement in pentylenetetrazole (PTZ)-induced seizure inrat and guinea pig. Peptides 18(7):1095-1097.

[0365] Krapivinsky, G., Gordon, E. A., Wickman B., Velimirovic, B.,Krapivinsky, L., Clapham, D. E. (1995) The G-protein-gated atrial K+channel IKACh is a heteromultimer of two inwardly rectifyingK(+)-channel proteins. Nature 374:135-141.

[0366] Krapivinsky, G., Krapivinsky, L., Velimirovic, B., Wickman, K.,Navarro, B., Clapham, D. E., (1995b) The cardiac inward rectifier K+channel subunit, CIR, does not comprise the ATP-sensitive K+ channel,IKATP. J. Biol. Chem. ZM: 28777-28779.

[0367] Kubo, Y., Reuveny, E., SlesInger, P. A., Jan, Y. N., Jan, L. Y.(1993) Primary structure and functional expression of a ratG-protein-coupled muscarinic potassium channel. Nature 364: 802-806.

[0368] Lazareno, S. and Birdsall N. J. M. (1993) Pharmacologicalcharacterization of acetylcholine stimulated [²⁵S]-GTPγS bindingmediated by human muscarinic m1-m4 receptors: antagonist studies. Br. J.Pharmacology, 109: 1120-1127.

[0369] Ludwig, D. S., Mountjoy, K. G., Tatro, J. B., Gillette, J. A.,Frederich, R. C., Flier, J. S., and Maratos-Flier, E. (1998)Melanin-concentrating hormone: a functional melanocortin antagonist inthe hypothalamus Am.J.Physiol.Endocrinol.Metab. 274(4):E627-E633.

[0370] MacKenzie, F. J., Hunter, A. J., Daly, C., Wilson, C. A. (1984)Evidence that the dopaminergic incerto-hypothalamic tract has astimulatory effect on ovulation and gonadotropin release.Neuroendocrinology 39:289-295.

[0371] Masu, Y. et al. (1994) Nature 329:21583-21586.

[0372] McBride, R. B., Beckwith, B. E., Swenson, R. R., Sawyer, T. K.,Hadley, M. E., Matsunaga, T. O. and Hruby, V. J. (1994) The actions ofmelanin-concentrating hormone (MCH) on passive avoidance in rats: Apreliminary study. Peptides 15:757-759.

[0373] Melki, J., Abdelhak, S., Sheth, P., Bachelot, M. F., Burlet, P.,Marcadet, A., Aicardi, J., Barois, A., Carriere, J. P., Fardeau, M.,Fontan, D., Ponsot, G., Billette, T., Angelini, C., Barbosa, C.,Ferriere, G., Lanzi, G., Ottolini, A., Babron, M. C., Cohen, D.,Hanauer, A., Clerget-Darpoux, G., Lathrop, M., Munnich, A. and Frezal,J. (1990) Gene for chronic proximal spinal muscular atrophies maps tochromosome 5q. Nature (London) 344:767-768.

[0374] Miller, C. L., Hruby, V., Matsubaga, T., Bickford, P. (1993)α-MSH and MCH are functional antagonists in a CNS auditory paradigm.Peptides 19:1-10.

[0375] Miller, J., Germain, R. N., Efficient cell surface expression ofclass II MHC molecules in the absence of associated invariant chain.J.Exp.Med. 164:1478-1489 (1986).

[0376] Morishita, F., Hashito, K., Fujimoto, M. and Yamada, K. (1993)Possible involvement of pertussis toxin-sensitive GTP-binding protein inthe α2-adrenoceptor-mediated melanosome-aggregation response of goldfishmelanophores. J. Exp. Zoology 266:173-180.

[0377] Nahon, J. L., Presse, F., Bittencourt, J. C., Sawchenko, P., andVale, W. (1989) The rat melanin-concentrating hormone mRNA encodesmultiple putative neuropeptides coexpressed in the dorsolateralhypothalamus. Endocrinology 125:2056-2065.

[0378] Nahon, J-L. (1994) The melanin-concentrating hormone: from thepeptide to the gene. Critical Rev. in Neurobiol 221:221-262.

[0379] Parkes, D. G. (1996) Diuretic and natriuretic actions of melaninconcentrating hormone in conscious sheep. J. Neuroendocrinol. 8:57-63.

[0380] Parkes, D. and Vale, W. (1993) Secretion of melanin-concentratinghormone and neuropeptide-EI from cultured rat hypothalamic cells.Endocrinology 131:1826-1831.

[0381] Pedeutour, F., Szpirer, C. and Nahon, J. L. (1994) Assignment ofthe human pro-melanin-concentrating hormone gene (PMCH) to chromosome12q23-24 and two variant genes (PMCHL1 and PMCHL2) to chromosome 5p14and 5q12-q13. Genomics 19:31-37.

[0382] Presse, F., Hervieu, G., Imaki, T., Sawchenko, P. E., Vale, W.,and Nahon, J-L. (1992) Rat melanin-concentrating hormone messengerribonucleic acid expression: marked changes during development and afterstress and glucocorticoid stimuli. Endocrinology 131:1241-1250.

[0383] Qu, D., Ludwig, D. S., Gammeltoft, S., Piper, M., Pelleymounter,M. A., Cullen, M. J., Foulds Mathes, W., Przypek, J., Kanarek, R. andMaratos-Flier, E. (1996) A role for melanin-concentrating hormone in thecentral regulation of feeding behaviour. Nature 380:243-247.

[0384] Qu, D., Mastaitis, J.W., Tritcs, N.A. and Maratos-Flier, E.(1998) 80^(th) Annual Meeting o_ the Encocrine Society in New Orleans.Abs. # P1-494.

[0385] Quick, M. W., Lester, H. A. Methods for expression ofexcitability proteins in Xenopus oocytes. Meth. Neurosci. 19:261-279(1994).

[0386] Rossi, M., Choi, S. J., O'Shea, D., Miyoshi, T., Ghatei, A. andBloom, S. R. (1997) Melanin-concentrating hormone acutely stimulatesfeeding, but chronic administration has no effect on body weight.Endocrinology 138:351-355.

[0387] Sahu, A. (1998) Evidence suggesting that galanin (GAL),melanin-concentrating hormone (MCH), neurotensin (NT),proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets ofleptin signaling in the hypothalamus. Endocrinology 139(2) :795-798.

[0388] Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M. et al., (1998) Orexins and orexin receptors: A family of hypothalamicneuropeptides and G protein-coupled receptors that regulate feedingbehavior. Cell 92:573-585.

[0389] Salon, J. A. and Owicki, J. C. (1995) Real-time measurements ofreceptor activity: Applications of microphysiometric techniques toreceptor biology. In: Methods in Neuroscience 25:201-223 (AcademicPress, 1995).

[0390] Sambrook, J., Fritsch, E. F., and Maniatis, T., In: MolecularCloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), 1989.

[0391] Sanchez, M., Baker, B. I. and Celis, M. (1997)Melanin-concentrating hormone (MCH) antagonizes the effects of α-MSH andneuropeptide E-I on grooming and locomotor activities in the rat.Peptides 18:393-396.

[0392] Schilling K., Luk, D., Morgan J., and Curran, T (1991) Regulationof a fos-lacZ fusion gene: A paradigm for quantitative analysis ofstimulus transcription coupling. Proc. Nat. Acad. Sci (USA)88:5665-5669.

[0393] Sherrington, R., Brynjolfsson, J., Petursson, H., Potter, M.,Dudleston, K., Barraclough, B., Wasmuth, J., Dobbs, M. and Gurling, H.(1988) Localization of a susceptibility locus for schizophrenia onchromosome 5. Nature (London) 33: 164-167.

[0394] Spurney, R. F.; Coffman, T. M. (1997) The C-terminus of thethromboxane receptor contributes to coupling and desensitization in amouse mesangial cell line. J. Pharmacol. Exp. Ther. 283(1):207-215 (Oct.1997).

[0395] Stuart, R. O., Sun, A., Bush, K. T., and Nigam, S. K. (1996)Dependence of epithelial intercellular junction biogenesis onthapsigargin-sensitive intracellular calcium stores. J Biol Chem (Jun.7, 1996) 271(23):13636-41.

[0396] Svenssson, S. P., Norberg, T., Anderssol, R. G., Grundstrom, N.and Karisson, J. O. G. (1991) MCH-induced piament aggregation in teleostmelanophores is associated with a cAMP reduction. Life Sci.48:2043-2046.

[0397] Takahashi, T., Neher, E., and Sakmann, B. (1987) Rat brainserotonin receptors in Xenopus oocytes are coupled by intracellularcalcium to endogenous channels. Proc Natl Acad Sci USA (1987 Jul)84(14):5063-7.

[0398] Tian, W., Duzic, E., Lanier, S., and Deth R. (1994) Determinantsof α-Adrenergic Receptor Activation of G protein: Evidence for aPrecoupled Receptor/G protein State. Molecular Pharmacology, 45:524-531.

[0399] Toumaniantz, G., Bittencourt, J. C., and Nahon, J. L. (1996) Therat melanin-concentrating hormone gene encodes an additional putativeprotein in a different reading frame. Endocrinology 137:4518-4521.

[0400] Twells, R., Weber, J., Orozco, G., Farrell, M., Williamson, R.and Chamberlain, S. (1992) Chromosomal assignment of the locus causingolivo-ponto-cerebellar atrophy (SCA2) in a cuban founder population.Cytogent. Cell. Cenet. 61:262-265.

[0401] Underwood, D. J., Strader, C. D., Rivero, R., Patchett, A. A.,Greenlee, W., and Prendergast, K. (1994) Structural model of antagonistand agonist binding to the anciotensin II, AT1 subtype, G proteincoupled receptor. Chem Biol (1994 Dec) 1(4):211-21.

[0402] Viale, A., Zhixing, Y., Breton, C., Pedeutour, F., Coquerel, A.,Jordan, D., Nahon, J. L. (1997) The melanin-concentratino hormone genein human: flanking region analysis, fine chromosome mapping, andtissue-specific expression. Mol. Brain Res. 46:243-255.

[0403] Westbrook, C. A., Neuman, W. L., McPherson, J., Camper, S.,Wasmuth, J., Plaetke, R. and Williamson, R. (1992) Report of the secondinternational workshop on human chromosome 5 mapping. Cytogenet. Cell.Genet. 61:225-231.

[0404] Yalkinoglu, A. C., Spreyer, P., Bechem, M., Apeler, N., andWohlfeil, S. (1995) Induction of c-fos expression in rat vascular smoothmuscle reporter cell by selective activation of the thrombin receptor.J. Receptor and Signal Transduction, 15(1-4) :117-130.

1 28 1 1269 DNA HOMO SAPIENS 1 atgtcagtgg gagccatgaa gaagggagtggggagggcag ttgggcttgg aggcggcagc 60 ggctgccagg ctacggagga agacccccttcccgactgcg gggcttgcgc tccgggacaa 120 ggtggcaggc gctggaggct gccgcagcctgcgtgggtgg aggggagctc agctcggttg 180 tgggagcagg cgaccggcac tggctggatggacctggaag cctcgctgct gcccactggt 240 cccaatgcca gcaacacctc tgatggccccgataacctca cttcagcagg atcacctcct 300 cgcacgggga gcatctccta catcaacatcatcatgcctt cggtgttcgg caccatctgc 360 ctcctgggca tcatcgggaa ctccacggtcatcttcgcgg tcgtgaagaa gtccaagctg 420 cactggtgca acaacgtccc cgacatcttcatcatcaacc tctcggtagt agatctcctc 480 tttctcctgg gcatgccctt catgatccaccagctcatgg gcaatggggt gtggcacttt 540 ggggagacca tgtgcaccct catcacggccatggatgcca atagtcagtt caccagcacc 600 tacatcctga ccgccatggc cattgaccgctacctggcca ctgtccaccc catctcttcc 660 acgaagttcc ggaagccctc tgtggccaccctggtgatct gcctcctgtg ggccctctcc 720 ttcatcagca tcacccctgt gtggctgtatgccagactca tccccttccc aggaggtgca 780 gtgggctgcg gcatacgcct gcccaacccagacactgacc tctactggtt caccctgtac 840 cagtttttcc tggcctttgc cctgccttttgtggtcatca cagccgcata cgtgaggatc 900 ctgcagcgca tgacgtcctc agtggcccccgcctcccagc gcagcatccg gctgcggaca 960 aagagggtga cccgcacagc catcgccatctgtctggtct tctttgtgtg ctgggcaccc 1020 tactatgtgc tacagctgac ccagttgtccatcagccgcc cgaccctcac ctttgtctac 1080 ttatacaatg cggccatcag cttgggctatgccaacagct gcctcaaccc ctttgtgtac 1140 atcgtgctct gtgagacgtt ccgcaaacgcttggtcctgt cggtgaagcc tgcagcccag 1200 gggcagcttc gcgctgtcag caacgctcagacggctgacg aggagaggac agaaagcaaa 1260 ggcacctga 1269 2 422 PRT HOMOSAPIENS 2 Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val GlyLeu 1 5 10 15 Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro LeuPro Asp 20 25 30 Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp ArgLeu Pro 35 40 45 Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp GluGln Ala 50 55 60 Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu ProThr Gly 65 70 75 80 Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn LeuThr Ser Ala 85 90 95 Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile AsnIle Ile Met 100 105 110 Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly IleIle Gly Asn Ser 115 120 125 Thr Val Ile Phe Ala Val Val Lys Lys Ser LysLeu His Trp Cys Asn 130 135 140 Asn Val Pro Asp Ile Phe Ile Ile Asn LeuSer Val Val Asp Leu Leu 145 150 155 160 Phe Leu Leu Gly Met Pro Phe MetIle His Gln Leu Met Gly Asn Gly 165 170 175 Val Trp His Phe Gly Glu ThrMet Cys Thr Leu Ile Thr Ala Met Asp 180 185 190 Ala Asn Ser Gln Phe ThrSer Thr Tyr Ile Leu Thr Ala Met Ala Ile 195 200 205 Asp Arg Tyr Leu AlaThr Val His Pro Ile Ser Ser Thr Lys Phe Arg 210 215 220 Lys Pro Ser ValAla Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser 225 230 235 240 Phe IleSer Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe 245 250 255 ProGly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr 260 265 270Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu 275 280285 Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met 290295 300 Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr305 310 315 320 Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val PhePhe Val 325 330 335 Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln LeuSer Ile Ser 340 345 350 Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn AlaAla Ile Ser Leu 355 360 365 Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe ValTyr Ile Val Leu Cys 370 375 380 Glu Thr Phe Arg Lys Arg Leu Val Leu SerVal Lys Pro Ala Ala Gln 385 390 395 400 Gly Gln Leu Arg Ala Val Ser AsnAla Gln Thr Ala Asp Glu Glu Arg 405 410 415 Thr Glu Ser Lys Gly Thr 4203 1214 DNA RATTUS NORVEGICUS 3 gcaggcgacc tgcaccggct gcatggatctgcaaacctcg ttgctgtcca ctggccccaa 60 tgccagcaac atctccgatg gccaggataatctcacattg ccggggtcac ctcctcgcac 120 agggagtgtc tcctacatca acatcattatgccttccgtg tttggtacca tctgtctcct 180 gggcatcgtg ggaaactcca cggtcatctttgctgtggtg aagaagtcca agctacactg 240 gtgcagcaac gtccccgaca tcttcatcatcaacctctct gtggtggatc tgctcttcct 300 gctgggcatg cctttcatga tccaccagctcatggggaac ggcgtctggc actttgggga 360 aaccatgtgc accctcatca cagccatggacgccaacagt cagttcacta gcacctacat 420 cctgactgcc atgaccattg accgctacttggccaccgtc caccccatct cctccaccaa 480 gttccggaag ccctccatgg ccaccctggtgatctgcctc ctgtgggcgc tctccttcat 540 cagtatcacc cctgtgtggc tctacgccaggctcattccc ttcccagggg gtgctgtggg 600 ctgtggcatc cgcctgccaa acccggacactgacctctac tggttcactc tgtaccagtt 660 tttcctggcc tttgcccttc cgtttgtggtcattaccgcc gcatacgtga aaatactaca 720 gcgcatgacg tcttcggtgg ccccagcctcccaacgcagc atccggcttc ggacaaagag 780 ggtgacccgc acggccattg ccatctgtctggtcttcttt gtgtgctggg caccctacta 840 tgtgctgcag ctgacccagc tgtccatcagccgcccgacc ctcacgtttg tctacttgta 900 caacgcggcc atcagcttgg gctatgctaacagctgcctg aacccctttg tgtacatagt 960 gctctgtgag acctttcgaa aacgcttggtgttgtcagtg aagcctgcag cccaggggca 1020 gctccgcacg gtcagcaacg ctcagacagctgatgaggag aggacagaaa gcaaaggcac 1080 ctgacaattc cccagtcgcc tccaagtcaggccaccccat caaaccgtgg ggagagatac 1140 tgagattaaa cccaaggcta ccctgggagaatgcagaggc tggaggctgg gggcttgtag 1200 caaccacatt ccac 1214 4 353 PRTRATTUS NORVEGICUS 4 Met Asp Leu Gln Thr Ser Leu Leu Ser Thr Gly Pro AsnAla Ser Asn 1 5 10 15 Ile Ser Asp Gly Gln Asp Asn Leu Thr Leu Pro GlySer Pro Pro Arg 20 25 30 Thr Gly Ser Val Ser Tyr Ile Asn Ile Ile Met ProSer Val Phe Gly 35 40 45 Thr Ile Cys Leu Leu Gly Ile Val Gly Asn Ser ThrVal Ile Phe Ala 50 55 60 Val Val Lys Lys Ser Lys Leu His Trp Cys Ser AsnVal Pro Asp Ile 65 70 75 80 Phe Ile Ile Asn Leu Ser Val Val Asp Leu LeuPhe Leu Leu Gly Met 85 90 95 Pro Phe Met Ile His Gln Leu Met Gly Asn GlyVal Trp His Phe Gly 100 105 110 Glu Thr Met Cys Thr Leu Ile Thr Ala MetAsp Ala Asn Ser Gln Phe 115 120 125 Thr Ser Thr Tyr Ile Leu Thr Ala MetThr Ile Asp Arg Tyr Leu Ala 130 135 140 Thr Val His Pro Ile Ser Ser ThrLys Phe Arg Lys Pro Ser Met Ala 145 150 155 160 Thr Leu Val Ile Cys LeuLeu Trp Ala Leu Ser Phe Ile Ser Ile Thr 165 170 175 Pro Val Trp Leu TyrAla Arg Leu Ile Pro Phe Pro Gly Gly Ala Val 180 185 190 Gly Cys Gly IleArg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe 195 200 205 Thr Leu TyrGln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile 210 215 220 Thr AlaAla Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala 225 230 235 240Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg 245 250255 Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr 260265 270 Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr275 280 285 Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala AsnSer 290 295 300 Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr PheArg Lys 305 310 315 320 Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln GlyGln Leu Arg Thr 325 330 335 Val Ser Asn Ala Gln Thr Ala Asp Glu Glu ArgThr Glu Ser Lys Gly 340 345 350 Thr 5 26 DNA ARTIFICIAL SEQUENCE PRIMER5 gggaactcca cggtcatctt cgcggt 26 6 26 DNA ARTIFICIAL SEQUENCE PRIMER 6tagcggtcaa tggccatggc ggtcag 26 7 45 DNA ARTIFICIAL SEQUENCE PROBE 7ctcctgggca tgcccttcat gatccaccag ctcatgggca atggg 45 8 25 DNA ARTIFICIALSEQUENCE PRIMER 8 cttctaggcc tgtacggaag tgtta 25 9 27 DNA ARTIFICIALSEQUENCE PRIMER 9 gttgtggttt gtccaaactc atcaatg 27 10 37 DNA ARTIFICIALSEQUENCE PRIMER 10 cgcggatcca ttatgtctgc actccgaagg aaatttg 37 11 38 DNAARTIFICIAL SEQUENCE PRIMER 11 cgcgaattct tatgtgaagc gatcagagtt catttttc38 12 34 DNA ARTIFICIAL SEQUENCE PRIMER 12 gcgggatccg ctatggctggtgattctagg aatg 34 13 29 DNA ARTIFICIAL SEQUENCE PRIMER 13 ccggaattcccctcacaccg agcccctgg 29 14 20 DNA ARTIFICIAL SEQUENCE PRIMER 14tcagctcggt tgtgggagca 20 15 18 DNA ARTIFICIAL SEQUENCE PRIMER 15cttggacttc ttcacgac 18 16 100 PRT ARTIFICIAL SEQUENCE MUTATION CLONE 16Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Thr Ala Val Gly Leu 1 5 1015 Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asp 20 2530 Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro 35 4045 Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala 50 5560 Thr Gly Thr Gly Trp Ala Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly 65 7075 80 Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala 8590 95 Gly Ser Pro Pro 100 17 100 PRT ARTIFICIAL SEQUENCE MUTATION CLONE17 Met Ser Val Gly Ala Ala Lys Lys Gly Val Gly Arg Ala Val Gly Leu 1 510 15 Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asp 2025 30 Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro 3540 45 Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala 5055 60 Thr Gly Thr Gly Trp Ala Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly 6570 75 80 Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala85 90 95 Gly Ser Pro Pro 100 18 31 DNA ARTIFICIAL SEQUENCE PRIMER 18cggcactggc tgggcggacc tggaagcctc g 31 19 31 DNA ARTIFICIAL SEQUENCEPRIMER 19 cgaggcttcc aggtccgccc agccagtgcc g 31 20 32 DNA ARTIFICIALSEQUENCE PRIMER 20 atgtcagtgg gagccgcgaa gaagggagtg gg 32 21 32 DNAARTIFICIAL SEQUENCE PRIMER 21 cccactccct tcttcgcggc tcccactgac at 32 2233 DNA ARTIFICIAL SEQUENCE PRIMER 22 taatgtgtct aggtggcgtc agtgggagccatg 33 23 33 DNA ARTIFICIAL SEQUENCE PRIMER 23 catggctccc actgacgccacctagacaca tta 33 24 37 DNA ARTIFICIAL SEQUENCE PRIMER 24 tgacactaagcttcactggc tggatggacc tggaagc 37 25 24 DNA ARTIFICIAL SEQUENCE PRIMER 25gcccaggaga aagaggagat ctac 24 26 422 PRT ARTIFICIAL SEQUENCE MUTATED MCHRECEPTOR 26 Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val GlyLeu 1 5 10 15 Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro LeuPro Asp 20 25 30 Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp ArgLeu Pro 35 40 45 Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp GluGln Ala 50 55 60 Thr Gly Thr Gly Trp Ala Asp Leu Glu Ala Ser Leu Leu ProThr Gly 65 70 75 80 Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn LeuThr Ser Ala 85 90 95 Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile AsnIle Ile Met 100 105 110 Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly IleIle Gly Asn Ser 115 120 125 Thr Val Ile Phe Ala Val Val Lys Lys Ser LysLeu His Trp Cys Asn 130 135 140 Asn Val Pro Asp Ile Phe Ile Ile Asn LeuSer Val Val Asp Leu Leu 145 150 155 160 Phe Leu Leu Gly Met Pro Phe MetIle His Gln Leu Met Gly Asn Gly 165 170 175 Val Trp His Phe Gly Glu ThrMet Cys Thr Leu Ile Thr Ala Met Asp 180 185 190 Ala Asn Ser Gln Phe ThrSer Thr Tyr Ile Leu Thr Ala Met Ala Ile 195 200 205 Asp Arg Tyr Leu AlaThr Val His Pro Ile Ser Ser Thr Lys Phe Arg 210 215 220 Lys Pro Ser ValAla Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser 225 230 235 240 Phe IleSer Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe 245 250 255 ProGly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr 260 265 270Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu 275 280285 Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met 290295 300 Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr305 310 315 320 Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val PhePhe Val 325 330 335 Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln LeuSer Ile Ser 340 345 350 Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn AlaAla Ile Ser Leu 355 360 365 Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe ValTyr Ile Val Leu Cys 370 375 380 Glu Thr Phe Arg Lys Arg Leu Val Leu SerVal Lys Pro Ala Ala Gln 385 390 395 400 Gly Gln Leu Arg Ala Val Ser AsnAla Gln Thr Ala Asp Glu Glu Arg 405 410 415 Thr Glu Ser Lys Gly Thr 42027 422 PRT ARTIFICIAL SEQUENCE MUTATED MCH RECEPTOR 27 Met Ser Val GlyAla Ala Lys Lys Gly Val Gly Arg Ala Val Gly Leu 1 5 10 15 Gly Gly GlySer Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asp 20 25 30 Cys Gly AlaCys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro 35 40 45 Gln Pro AlaTrp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala 50 55 60 Thr Gly ThrGly Trp Ala Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly 65 70 75 80 Pro AsnAla Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala 85 90 95 Gly SerPro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met 100 105 110 ProSer Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser 115 120 125Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn 130 135140 Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu 145150 155 160 Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly AsnGly 165 170 175 Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr AlaMet Asp 180 185 190 Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr AlaMet Ala Ile 195 200 205 Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser SerThr Lys Phe Arg 210 215 220 Lys Pro Ser Val Ala Thr Leu Val Ile Cys LeuLeu Trp Ala Leu Ser 225 230 235 240 Phe Ile Ser Ile Thr Pro Val Trp LeuTyr Ala Arg Leu Ile Pro Phe 245 250 255 Pro Gly Gly Ala Val Gly Cys GlyIle Arg Leu Pro Asn Pro Asp Thr 260 265 270 Asp Leu Tyr Trp Phe Thr LeuTyr Gln Phe Phe Leu Ala Phe Ala Leu 275 280 285 Pro Phe Val Val Ile ThrAla Ala Tyr Val Arg Ile Leu Gln Arg Met 290 295 300 Thr Ser Ser Val AlaPro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr 305 310 315 320 Lys Arg ValThr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val 325 330 335 Cys TrpAla Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser 340 345 350 ArgPro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu 355 360 365Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys 370 375380 Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln 385390 395 400 Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala Asp Glu GluArg 405 410 415 Thr Glu Ser Lys Gly Thr 420 28 353 PRT ARTIFICIALSEQUENCE MUTATED MCH RECEPTOR 28 Met Asp Leu Glu Ala Ser Leu Leu Pro ThrGly Pro Asn Ala Ser Asn 1 5 10 15 Thr Ser Asp Gly Pro Asp Asn Leu ThrSer Ala Gly Ser Pro Pro Arg 20 25 30 Thr Gly Ser Ile Ser Tyr Ile Asn IleIle Met Pro Ser Val Phe Gly 35 40 45 Thr Ile Cys Leu Leu Gly Ile Ile GlyAsn Ser Thr Val Ile Phe Ala 50 55 60 Val Val Lys Lys Ser Lys Leu His TrpCys Asn Asn Val Pro Asp Ile 65 70 75 80 Phe Ile Ile Asn Leu Ser Val ValAsp Leu Leu Phe Leu Leu Gly Met 85 90 95 Pro Phe Met Ile His Gln Leu MetGly Asn Gly Val Trp His Phe Gly 100 105 110 Glu Thr Met Cys Thr Leu IleThr Ala Met Asp Ala Asn Ser Gln Phe 115 120 125 Thr Ser Thr Tyr Ile LeuThr Ala Met Ala Ile Asp Arg Tyr Leu Ala 130 135 140 Thr Val His Pro IleSer Ser Thr Lys Phe Arg Lys Pro Ser Val Ala 145 150 155 160 Thr Leu ValIle Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr 165 170 175 Pro ValTrp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val 180 185 190 GlyCys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe 195 200 205Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile 210 215220 Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala 225230 235 240 Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val ThrArg 245 250 255 Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp AlaPro Tyr 260 265 270 Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg ProThr Leu Thr 275 280 285 Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu GlyTyr Ala Asn Ser 290 295 300 Cys Leu Asn Pro Phe Val Tyr Ile Val Leu CysGlu Thr Phe Arg Lys 305 310 315 320 Arg Leu Val Leu Ser Val Lys Pro AlaAla Gln Gly Gln Leu Arg Ala 325 330 335 Val Ser Asn Ala Gln Thr Ala AspGlu Glu Arg Thr Glu Ser Lys Gly 340 345 350 Thr

What is claimed is:
 1. An isolated nucleic acid encoding a human MCH1receptor or a mutant of such human MCH1 receptor which is activated byMCH or an analog or homolog thereof.
 2. The nucleic acid of claim 1,wherein the nucleic acid is DNA.
 3. The DNA of claim 2, wherein the DNAis cDNA.
 4. The DNA of claim 2, wherein the DNA is genomic DNA.
 5. Thenucleic acid of claim 1, wherein the nucleic acid is RNA.
 6. The nucleicacid of claim 1, wherein the human MCH1 receptor has an amino acidsequence identical to that encoded by the plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197).
 7. The nucleic acid of claim 1, wherein the humanMCH1 receptor comprises an amino acid sequence as shown in FIG. 2 (SEQID NO: 2).
 8. The nucleic acid of claim 1, wherein the mutant human MCH1receptor comprises an amino acid sequence as shown in FIG. 13 (SEQ IDNO: 27).
 9. The nucleic acid of claim 1, wherein the mutant human MCH1receptor comprises an amino acid sequence as shown in FIG. 14 (SEQ IDNO: 28).
 10. The nucleic acid of claim 1, wherein the mutant human MCH1receptor comprises an amino acid sequence as shown in FIG. 15 (SEQ IDNO: 29).
 11. A purified human MCH1 receptor protein.
 12. A vectorcomprising the nucleic acid of claim
 1. 13. The vector of claim 12adapted for expression in a cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the cell operativelylinked to the nucleic acid encoding the receptor so as to permitexpression thereof, wherein the cell is a bacterial, amphibian, yeast,insect or mammalian cell.
 14. The vector of claim 13, wherein the vectoris a baculovirus.
 15. The vector of claim 12, wherein the vector is aplasmid.
 16. The plasmid of claim 15 designated pEXJ.HR-TL231 (ATCCAccession No. 203197).
 17. A cell comprising the vector of claim
 13. 18.A cell of claim 17, wherein the cell is a non-mammalian cell.
 19. A cellof claim 18, wherein the non-mammalian cell is a Xenopus oocyte cell ora Xenopus melanophore cell.
 20. A cell of claim 17, wherein the cell isa mammalian cell.
 21. A mammalian cell of claim 20, wherein the cell isa COS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, aLM(tk-) cell, a mouse Y1 cell, or a CHO cell.
 22. An insect cellcomprising the vector of claim
 13. 23. An insect cell of claim 22,wherein the insect cell is an Sf9 cell, an Sf21 cell or a Trichoplusiani 5B-4 cell.
 24. A membrane preparation isolated from the cell of claim17.
 25. A nucleic acid probe comprising at least 15 nucleotides whichspecifically hybridizes with a nucleic acid encoding a human MCH1receptor, wherein the probe has a unique sequence corresponding to asequence present within one of the two strands of the nucleic acidencoding a human MCH1 receptor present in plasmid pEXJ.HR-T231 (ATCCAccession No. 203197).
 26. A nucleic acid probe comprising at least 15nucleotides which specifically hybridizes with a nucleic acid encoding ahuman MCH1 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO: 1) or (b) the reverse complement thereof.27. The nucleic acid probe of claim 25 or 26, wherein the nucleic acidis DNA.
 28. The nucleic acid probe of claim 25 or 26, wherein thenucleic acid is RNA.
 29. An antisense oligonucleotide having a sequencecapable of specifically hybridizing to the RNA of claim 5, so as toprevent translation of the RNA.
 30. An antisense oligonucleotide havinga sequence capable of specifically hybridizing to the genomic DNA ofclaim
 4. 31. An antisense oligonucleotide of claim 29 or 30, wherein theoligonucleotide comprises chemically modified nucleotides or nucleotideanalogues.
 32. An antibody capable of binding to a human MCH1 receptorencoded by the nucleic acid of claim
 1. 33. An agent capable ofcompetitively inhibiting the binding of the antibody of claim 32 to ahuman MCH1 receptor.
 34. An antibody of claim 32, wherein the antibodyis a monoclonal antibody or antisera.
 35. A pharmaceutical compositioncomprising (a) an amount of the oligonucleotide of claim 29 capable ofpassing through a cell membrane and effective to reduce expression of ahuman MCH1 receptor and (b) a pharmaceutically acceptable carriercapable of passing through the cell membrane.
 36. A pharmaceuticalcomposition of claim 35, wherein the oligonucleotide is coupled to asubstance which inactivates mRNA.
 37. A pharmaceutical composition ofclaim 36, wherein the substance which inactivates mRNA is a ribozyme.38. A pharmaceutical composition of claim 35, wherein thepharmaceutically acceptable carrier comprises a structure which binds toa human MCH1 receptor on a cell capable of being taken up by the cellsafter binding to the structure.
 39. A pharmaceutical composition ofclaim 35, wherein the pharmaceutically acceptable carrier is capable ofbinding to a human MCH1 receptor which is specific for a selected celltype.
 40. A pharmaceutical composition which comprises an amount of theantibody of claim 32 effective to block binding of a ligand to a humanMCH1 receptor and a pharmaceutically acceptable carrier.
 41. Atransgenic, nonhuman mammal expressing DNA encoding a human MCH1receptor of claim
 1. 42. A transgenic, nonhuman mammal comprising ahomologous recombination knockout of the native human MCH1 receptor. 43.A transgenic, nonhuman mammal whose genome comprises antisense DNAcomplementary to the DNA encoding a human MCH1 receptor of claim 1 soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the human MCH1 receptor and whichhybridizes to mRNA encoding the human MCH1 receptor, thereby reducingits translation.
 44. The transgenic, nonhuman mammal of claim 41 or 42,wherein the DNA encoding the human MCH1 receptor additionally comprisesan inducible promoter.
 45. The transgenic, nonhuman mammal of claim 41or 42, wherein the DNA encoding the human MCH1 receptor additionallycomprises tissue specific regulatory elements.
 46. A transgenic,nonhuman mammal of claim 41, 42, or 43, wherein the transgenic, nonhumanmammal is a mouse.
 47. A process for identifying a chemical compoundwhich specifically binds to a mammalian MCH1 receptor which comprisescontacting cells comprising DNA encoding, and expressing on their cellsurface, the mammalian MCH1 receptor, with the compound under conditionssuitable for binding, and detecting specific binding of the chemicalcompound to the mammalian MCH1 receptor, wherein the cells do notnormally express the mammalian MCH1 receptor and the DNA encoding themammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (SEQ ID NO: 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.
 48. A process for identifying achemical compound which specifically binds to a mammalian MCH1 receptorwhich comprises contacting a membrane preparation from cells comprisingDNA encoding, and expressing on their cell surface, the mammalian MCH1receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianMCH1 receptor, wherein the cells do not normally express the mammalianMCH1 receptor and the DNA encoding the mammalian MCH1 receptor (a)hybridizes to a nucleic acid having the defined sequence shown in FIG. 1(SEQ ID NO: 1) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a MCH1 ligand isadded to the culture and the CHO cells contain the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 49. The process of claim 47 or 48, wherein the mammalianMCH1 receptor is a human MCH1 receptor.
 50. The process of claim 47 or48, wherein the mammalian. MCH1 receptor is a rat MCH1 receptor.
 51. Theprocess of claim 47 or 48, wherein the mammalian MCH1 receptor hassubstantially the same amino acid sequence as the sequence of the humanMCH1 receptor encoded by plasmid pEXJ.HR-TL231 (ATCC Accession No.203197).
 52. The process of claim 47 or 48, wherein the mammalian MCH1receptor comprises substantially the same amino acid sequence as thatshown in FIG. 2 (SEQ ID NO: 2).
 53. The process of claim 47 or 48,wherein the mammalian MCH1 receptor comprises the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 2).
 54. The process of claim 47 or 48,wherein the mammalian MCH1 receptor comprises the amino acid sequenceshown in FIG. 13 (SEQ ID NO: 27).
 55. The process of claim 47 or 48,wherein the mammalian MCH1 receptor comprises the amino acid sequenceshown in FIG. 14 (SEQ ID NO: 28).
 56. The process of claim 47 or 48,wherein the mammalian MCH1 receptor comprises the amino acid sequenceshown in FIG. 15 (SEQ ID NO: 29).
 57. The process of claim 47 or 48,wherein the compound is not previously known to bind to a mammalian MCH1receptor.
 58. A compound identified by the process of claim
 57. 59. Aprocess of claim 47 or 48, wherein the cell is an insect cell.
 60. Theprocess of claim 47 or 48, wherein the cell is a mammalian cell.
 61. Theprocess of claim 60, wherein the cell is nonneuronal in origin.
 62. Theprocess if claim 61, wherein the nonneuronal cell is a COS-7 cell, 293human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1cell, or a LM(tk-) cell.
 63. A process of claim 60, wherein the compoundis a compound not previously known to bind to a mammalian MCH1 receptor.64. A compound identified by the process of claim
 63. 65. A processinvolving competitive binding for identifying a chemical compound whichspecifically binds to a mammalian MCH1 receptor which comprisescontacting cells expressing on their cell surface the mammalian MCH1receptor, with both the chemical compound and a second chemical compoundknown to bind to the receptor, and separately with only the secondchemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian MCH1 receptor, a decrease in the binding of the secondchemical compound to the mammalian MCH1 receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian MCH1 receptor, wherein the cells do not normally express themammalian MCH1 receptor and the DNA encoding the mammalian MCH1 receptor(a) hybridizes to a nucleic acid having the defined sequence shown inFIG. 1 (SEQ ID NO: 1) under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a MCH1 ligand isadded to the culture and the CHO cells contain the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 66. A process involving competitive binding for identifyinga chemical compound which specifically binds to a mammalian MCH1receptor which comprises contacting a membrane preparation from cellsexpressing on their cell surface the mammalian MCH1 receptor, with boththe chemical compound and a second chemical compound known to bind tothe receptor, and separately with only the second chemical compound,under conditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the mammalian MCH1receptor, a decrease in the binding of the second chemical compound tothe mammalian MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian MCH1receptor, wherein the cells do not normally express the mammalian MCH1receptor and the DNA encoding the mammalian MCH1 receptor (a) hybridizesto a nucleic acid having the defined sequence shown in FIG. 1 (SEQ IDNO: 1) under low stringency conditions or a sequence complementarythereto and (b) is further characterized by its ability to cause achange in the pH of a culture of CHO cells when a MCH1 ligand is addedto the culture and the CHO cells contain the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.
 67. A process of claim 65 or 66, wherein the mammalian MCH1receptor is a human MCH1 receptor or a mutant of such human MCH1receptor which is activated by MCH or an analog or homolog thereof. 68.A process of claim 65 or 66, wherein the mammalian MCH1 receptor is arat MCH1 receptor.
 69. The process of claim 65 or 66, wherein the cellis an insect cell.
 70. The process of claim 65 or 66, wherein the cellis a mammalian cell.
 71. The process of claim 70, wherein the cell isnonneuronal in origin.
 72. The process of claim 71, wherein thenonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHOcell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-) cell.
 73. Theprocess of claim 70, wherein the compound is not previously known tobind to a mammalian MCH1 receptor.
 74. A compound identified by theprocess of claim
 73. 75. A method of screening a plurality of chemicalcompounds not known to bind to a mammalian MCH1 receptor to identify acompound which specifically binds to the mammalian MCH1 receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian MCH1 receptor with the plurality of compounds notknown to bind specifically to the mammalian MCH1 receptor, underconditions permitting binding of compounds known to bind the mammalianMCH1 receptor; (b) determining whether the binding of a compound knownto bind to the mammalian MCH1 receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (c)separately determining the binding to the mammalian MCH1 receptor ocompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian MCH1receptor.
 76. A method of screening a plurality of chemical compoundsnot known to bind to a mammalian MCH1 receptor to identify a compoundwhich specifically binds to the mammalian MCH1 receptor, which comprises(a) contacting a membrane preparation from cells transfected with andexpressing DNA encoding the mammalian MCH1 receptor with the pluralityof compounds not known to bind specifically to the mammalian MCH1receptor under conditions permitting binding of compounds known to bindthe mammalian MCH1 receptor; (b) determining whether the binding of acompound known to bind to the mammalian MCH1 receptor is reduced in thepresence of the compounds within the plurality of compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so (c) separately determining the binding to themammalian MCH1 receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian MCH1 receptor.
 77. A method of claim 75 or 76,wherein the mammalian MCH1 receptor is a human MCH1 receptor or a mutantof such human MCH1 receptor which is activated by MCH or an analog orhomolog thereof.
 78. A method of claim 75 or 76, wherein the mammalianMCH1 receptor is a rat MCH1 receptor.
 79. A method of claim 75 or 76,wherein the cell is a mammalian cell.
 80. A method of claim 79, whereinthe mammalian cell is non-neuronal in origin.
 81. The method of claim80, wherein the non-neuronal cell is a COS-7 cell, a 293 human embryonickidney cell, a LM(tk-) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3cell.
 82. A method of detecting expression of a mammalian MCH1 receptorby detecting the presence of mRNA coding for the mammalian MCH1 receptorwhich comprises obtaining total mRNA from the cell and contacting themRNA so obtained with the nucleic acid probe of any of claims 25, 26,27, or 28 under hybridizing conditions, detecting the presence of mRNAhybridizing to the probe, and thereby detecting the expression of themammalian MCH1 receptor by the cell.
 83. A method of detecting thepresence of a mammalian MCH1 receptor on the surface of a cell whichcomprises contacting the cell with the antibody of claim 32 underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of the mammalian MCH1 receptor on the surface of the cell.84. A method of determining the physiological effects of varying levelsof activity of human MCH1 receptors which comprises producing atransgenic, nonhuman mammal of claim 44 whose levels of human MCH1receptor activity are varied by use of an inducible promoter whichregulates human MCH1 receptor expression.
 85. A method of determiningthe physiological effects of varying levels of activity of human MCH1receptors which comprises producing a panel of transgenic, nonhumanmammals of claim 44, each expressing a different amount of human MCH1receptor.
 86. A method for identifying an antagonist capable ofalleviating an abnormality, wherein the abnormality is alleviated bydecreasing the activity of a human MCH1 receptor comprisingadministering a compound to the transgenic, nonhuman mammal of claim 41,44, 45, or 46, and determining whether the compound alleviates thephysical and behavioral abnormalities displayed by the transgenic,nonhuman mammal as a result of overactivity of a human MCH1 receptor,the alleviation of the abnormality identifying the compound as anantagonist.
 87. An antagonist identified by the method of claim
 86. 88.A pharmaceutical composition comprising an antagonist of claim 87 and apharmaceutically acceptable carrier.
 89. A method of treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a human MCH1 receptor which comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition of claim 88, thereby treating the abnormality.
 90. A methodfor identifying an agonist capable of alleviating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a human MCH1 receptor comprising administering a compound to thetransgenic, nonhuman mammal of claim 41, 44, 45, or 46, and determiningwhether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.91. An agonist identified by the method of claim
 90. 92. Apharmaceutical composition comprising an agonist of claim 91 and apharmaceutically acceptable carrier.
 93. A method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a human MCH1 receptor which comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition of claim 92, thereby treating the abnormality.
 94. A methodfor diagnosing a predisposition to a disorder associated with theactivity of a specific mammalian allele which comprises: (a) obtainingDNA of subjects suffering from the disorder; (b) performing arestriction digest of the DNA with a panel of restriction enzymes; (c)electrophoretically separating the resulting DNA fragments on a sizinggel; (d) contacting the resulting gel with a nucleic acid probe capableof specifically hybridizing with a unique sequence included within thesequence of a nucleic acid molecule encoding a human MCH1 receptor andlabeled with a detectable marker; (e) detecting labeled bands which havehybridized to the DNA encoding a human MCH1 receptor of claim 1 labeledwith a detectable marker to create a unique band pattern specific to theDNA of subjects suffering from the disorder; (f) preparing DNA obtainedfor diagnosis by steps (a)-(e); and (g) comparing the unique bandpattern specific to the DNA of subjects suffering from the disorder fromstep (e) and the DNA obtained for diagnosis from step (f) to determinewhether the patterns are the same or different and to diagnose therebypredisposition to the disorder if the patterns are the same.
 95. Themethod of claim 94, wherein a disorder associated with the activity of aspecific mammalian allele is diagnosed.
 96. A method of preparing thepurified human MCH1 receptor of claim 11 which comprises: (a) inducingcells to express the human MCH1 receptor; (b) recovering the human, MCH1receptor from the induced cells; and (c) purifying the human MCH1receptor so recovered.
 97. A method of preparing the purified human MCH1receptor of claim 11 which comprises: (a) inserting nucleic acidencoding the human MCH1 receptor in a suitable vector; (b) introducingthe resulting vector in a suitable host cell; (c) placing the resultingcell in suitable condition permitting the production of the isolatedhuman MCH1 receptor; (d) recovering the human MCH1 receptor produced bythe resulting cell; and (e) purifying the human MCH1 receptor sorecovered.
 98. A process for determining whether a chemical compound isa mammalian MCH1 receptor agonist which comprises contacting cellstransfected with and expressing DNA encoding the mammalian MCH1 receptorwith the compound under conditions permitting the activation of themammalian MCH1 receptor, and detecting an increase in mammalian MCH1receptor activity, so as to thereby determine whether the compound is amammalian MCH1 receptor agonist.
 99. A process for determining whether achemical compound is a mammalian MCH1 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian MCH1 receptor with the compound in the presence of a knownmammalian MCH1 receptor agonist, under conditions permitting theactivation of the mammalian MCH1 receptor, and detecting a decrease inmammalian MCH1 receptor activity, so as to thereby determine whether thecompound is a mammalian MCH1 receptor antagonist.
 100. A process ofclaim 98 or 99, wherein the mammalian MCH1 receptor is a human MCH1receptor or a mutant of such human MCH1 receptor which is activated byMCH or an analog or homolog thereof.
 101. A process of claim 98 or 99,wherein the mammalian MCH1 receptor is a rat MCH1 receptor.
 102. Apharmaceutical composition which comprises an amount of a mammalian MCH1receptor agonist determined by the process of claim 98 effective toincrease activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.
 103. A pharmaceutical composition of claim 102,wherein the mammalian MCH1 receptor agonist is not previously known.104. A pharmaceutical composition which comprises an amount of amammalian MCH1 receptor antagonist determined by the process of claim 99effective to reduce activity of a mammalian MCH1 receptor and apharmaceutically acceptable carrier.
 105. A pharmaceutical compositionof claim 104, wherein the mammalian MCH1 receptor antagonist is notpreviously known.
 106. A process for determining whether a chemicalcompound specifically binds to and activates a mammalian MCH1 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the mammalian MCH1 receptor,wherein such cells do not normally express the mammalian MCH1 receptor,with the chemical compound under conditions suitable for activation ofthe mammalian MCH1 receptor, and measuring the second messenger responsein the presence and in the absence -of the chemical compound, a changein the second messenger response in the presence of the chemicalcompound indicating that the compound activates the mammalian MCH1receptor.
 107. The process of claim 106, wherein the second messengerresponse comprises chloride channel activation and the change in secondmessenger is an increase in the level of inward chloride current.
 108. Aprocess for determining whether a chemical compound specifically bindsto and inhibits activation of a mammalian MCH1 receptor, which comprisesseparately contacting cells producing a second messenger response andexpressing on their cell surface the mammalian MCH1 receptor, whereinsuch cells do not normally express the mammalian MCH1 receptor, withboth the chemical compound and a second chemical compound known toactivate the mammalian MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the mammalian MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian MCH1 receptor.
 109. The process of claim108, wherein the second messenger response comprises chloride channelactivation and the change in second messenger response is a smallerincrease in the level of inward chloride current in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound.
 110. A process of any ofclaims 106, 107, 108, or 109, wherein the mammalian MCH1 receptor is ahuman MCH1 receptor or a mutant of such human MCH1 receptor which isactivated by MCH or an analog or homolog thereof.
 111. A process of anyof claims 106, 107, 108, or 109, wherein the mammalian MCH1 receptor isa rat MCH1 receptor.
 112. The process of any of claims 106, 107, 108,109, or 110, wherein the cell is an insect cell.
 113. The process of anyof claims 106, 107, 108, 109, or 110, wherein the cell is a mammaliancell.
 114. The process of claim 113, wherein the mammalian cell isnonneuronal in origin.
 115. The process of claim 114, wherein thenonneuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidneycell, NIH-3T3 cell or LM(tk-) cell.
 116. The process of claim 106, 107,108, or 109, wherein the compound is not previously known to bind to amammalian MCH1 receptor.
 117. A compound determined by the process ofclaim
 116. 118. A pharmaceutical composition which comprises an amountof a mammalian MCH1 receptor agonist determined by the process of claim106 or 107 effective to increase activity of a mammalian MCH1 receptorand a pharmaceutically acceptable carrier.
 119. A pharmaceuticalcomposition of claim 118, wherein the mammalian MCH1 receptor agonist isnot previously known.
 120. A pharmaceutical composition which comprisesan amount of a mammalian MCH1 receptor antagonist determined by theprocess of claim 108 or 109 effective to reduce activity of a mammalianMCH1 receptor and a pharmaceutically acceptable carrier.
 121. Apharmaceutical composition of claim 120, wherein the mammalian MCH1receptor antagonist is not previously known.
 122. A method of screeninga plurality of chemical compounds not known to activate a mammalian MCH1receptor to identify a compound which activates the mammalian MCH1receptor which comprises: (a) contacting cells transfected with andexpressing the mammalian MCH1 receptor with the plurality of compoundsnot known to activate the mammalian MCH1 receptor, under conditionspermitting activation of the mammalian MCH1 receptor; (b) determiningwhether the activity of the mammalian MCH1 receptor is increased in thepresence of the compounds; and if so (c) separately determining whetherthe activation of the mammalian MCH1 receptor is increased by eachcompound included in the plurality of compounds, so as to therebyidentify the compound which activates the mammalian MCH1 receptor. 123.A method of claim 122, wherein the mammalian MCH1 receptor is a humanMCH1 receptor or a mutant of such human MCH1 receptor which is activatedby MCH or an analog or homolog thereof.
 124. A method of claim 122,wherein the mammalian MCH1 receptor is a rat MCH1 receptor.
 125. Amethod of screening a plurality of chemical compounds not known toinhibit the activation of a mammalian MCH1 receptor to identify acompound which inhibits the activation of the mammalian MCH1 receptor,which comprises: (a) contacting cells transfected with and expressingthe mammalian MCH1 receptor with the plurality of compounds in thepresence of a known mammalian MCH1 receptor agonist, under conditionspermitting activation of the mammalian MCH1 receptor; (b) determiningwhether the activation of the mammalian MCH1 receptor is reduced in thepresence of the plurality of compounds, relative to the activation ofthe mammalian MCH1 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the mammalian MCH1 receptor for each compound included inthe plurality of compounds, so as to thereby identify the compound whichinhibits the activation of the mammalian MCH1 receptor.
 126. A method ofclaim 125, wherein the mammalian MCH1 receptor is a human MCH1 receptoror a mutant of such human MCH1 receptor which is activated by MCH or ananalog or homolog thereof.
 127. A method of claim 125, wherein themammalian MCH1 receptor is a rat MCH1 receptor.
 128. A method of any ofclaims 123, 124, 125, 126, or 127, wherein the cell is a mammalian cell.129. A method of claim 128, wherein the mammalian cell is non-neuronalin origin.
 130. The method of claim 129, wherein the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or anNIH-3T3 cell.
 131. A pharmaceutical composition comprising a compoundidentified by the method of claim 123 or 124 effective to increasemammalian MCH1 receptor activity and a pharmaceutically acceptablecarrier.
 132. A pharmaceutical composition comprising a compoundidentified by the method of claim 125 or 126 effective to decreasemammalian MCH1 receptor activity and a pharmaceutically acceptablecarrier.
 133. A method of treating an abnormality in a subject whereinthe abnormality is alleviated by increasing the activity of a mammalianMCH1 receptor which comprises administering to the subject an amount ofa compound which is a mammalian MCH1 receptor agonist effective to treatthe abnormality.
 134. A method of claim 133, wherein the abnormality isa regulation of a steroid or pituitary hormone disorder, an epinephrinerelease disorder, a gastrointestinal disorder, a cardiovasculardisorder, an electrolyte balance disorder, hypertension, diabetes, arespiratory disorder, asthma, a reproductive function disorder, animmune disorder, an endocrine disorder, a musculoskeletal disorder, aneuroendocrine disorder, a cognitive disorder, a memory disorder, asensory modulation and transmission disorder, a motor coordinationdisorder, a sensory integration disorder, a motor integration disorder,a dopaminergic function disorder, a sensory transmission disorder, anolfaction disorder, a sympathetic innervation disorder, pain, psychoticbehavior, morphine tolerance, opiate addiction, an affective disorder, astress-related disorder, a fluid-balance disorder, a seizure disorder,or migraine.
 135. A method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian MCH1 receptor which comprises administering to the subject anamount of a compound which is a mammalian MCH1 receptor antagonisteffective to treat the abnormality.
 136. A method of claim 135, whereinthe abnormality is a regulation of a steroid or pituitary hormonedisorder, an epinephrine release disorder, a gastrointestinal disorder,a cardiovascular disorder, an electrolyte balance disorder,hypertension, diabetes, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, a sensory modulation and transmissiondisorder, a motor coordination disorder, a sensory integration disorder,a motor integration disorder, a dopaminergic function disorder, asensory transmission disorder, an olfaction disorder, a sympatheticinnervation disorder, pain, psychotic behavior, morphine tolerance,opiate addiction, an affective disorder, a stress-related disorder, afluid-balance disorder, a seizure disorder, or migraine.
 137. A processfor making a composition of matter which specifically binds to amammalian MCH1 receptor which comprises identifying a chemical compoundusing the process of any of claims 47, 48, 65, 66, 75, or 76 and thensynthesizing the chemical compound or a novel structural and functionalanalog or homolog thereof.
 138. A process for making a composition ofmatter which specifically binds to a mammalian MCH1 receptor whichcomprises identifying a chemical compound using the process of any ofclaims 98, 106, or 122 and then synthesizing the chemical compound or anovel structural and functional analog or homolog thereof.
 139. Aprocess for making a composition of matter which specifically binds to amammalian MCH1 receptor which comprises identifying a chemical compoundusing the process of any of claims 99, 108, or 125 and then synthesizingthe chemical compound or a novel structural and functional analog orhomolog thereof.
 140. The process of any of claims 137, 138, or 139,wherein the mammalian MCH1 receptor is a human MCH1 receptor or a mutantof such human MCH1 receptor which is activated by MCH or an analog orhomolog thereof.
 141. The process of any of claims 137, 138, or 139,wherein the mammalian MCH1 receptor is a human MCH1 receptor.
 142. Aprocess for preparing a composition which comprises admixing apharmaceutically acceptable carrier and a therapeutically effectiveamount of a chemical compound identified by the process of any of claims47, 48, 65, 66, 75, or 76 or a novel structural and functional analog orhomolog thereof.
 143. A process for preparing a composition whichcomprises admixing a pharmaceutically acceptable carrier and atherapeutically effective amount of a chemical compound identified bythe process of any of claims 98, 106, or 122 or a novel structural andfunctional analog or homolog thereof.
 144. A process for preparing acomposition which comprises admixing a pharmaceutically acceptablecarrier and a therapeutically effective amount of a chemical compoundidentified by the process of any of claims 99, 108, or 125 or a novelstructural and functional analog or homolog thereof.
 145. The process ofany of claims 142, 143, or 144, wherein the mammalian MCH1 receptor is ahuman MCH1 receptor or a mutant of such human MCH1 receptor which isactivated by MCH or an analog or homolog thereof.
 146. The process ofany of claims 142, 143, or 144, wherein the mammalian MCH1 receptor is arat MCH1 receptor.
 147. A process for determining whether a chemicalcompound is a human MCH1 receptor antagonist which comprises contactingcells transfected with and expressing DNA encoding the human MCH1receptor with the compound in the presence of a known human MCH1receptor agonist, under conditions permitting the activation of thehuman MCH1 receptor, and detecting a decrease in human MCH1 receptoractivity, so as to thereby determine whether the compound is a humanMCH1 receptor antagonist, wherein the DNA encoding the human MCH1receptor comprises the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH,and the cells do not express the MCH1 receptor prior to transfectingthem.
 148. A process for determining whether a chemical compoundspecifically binds to and inhibits activation of a human MCH1 receptor,which comprises separately contacting cells expressing on their cellsurface the human MCH1 receptor and producing a second messengerresponse upon activation of the human MCH1 receptor, wherein such cellsdo not normally express the human MCH1 receptor and the DNA encoding thehuman MCH1 receptor comprises the sequence shown in FIG. 1 (Seq IDNo. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No.203197), with both the chemical compound and a second chemical compoundknown to activate the human MCH1 receptor, and with only the secondchemical compound, under conditions suitable for activation of the humanMCH1 receptor, and measuring the second messenger response in thepresence of only the second chemical compound and in the presence ofboth the second chemical compound and the chemical compound, a smallerchange in the second messenger response in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound indicating that the chemicalcompound inhibits activation of the human MCH1 receptor, wherein thesecond chemical compound is MCH or a homolog or analog of MCH.
 149. Theprocess of claim 148, wherein the second messenger response compriseschloride channel activation and the change in second messenger responseis a smaller increase in the level of inward chloride current in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound.
 150. A methodof screening a plurality of chemical compounds not known to inhibit theactivation of a human MCH1 receptor to identify a compound whichinhibits the activation of the human MCH1 receptor, which comprises: (a)contacting cells transfected with and expressing the human MCH1receptor, wherein such cells do not normally express the human MCH1receptor and the DNA encoding the human MCH1 receptor comprises thesequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), with the plurality ofcompounds in the presence of a known human MCH1 receptor agonist, underconditions permitting activation of the human MCH1 receptor, wherein theknown MCH1 receptor agonist is MCH or a homolog or analog of MCH; (b)determining whether the activation of the human MCH1 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the human MCH1 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the extent of inhibitionof activation of the human MCH1 receptor for each compound included inthe plurality of compounds, so as to thereby identify the compound whichinhibits the activation of the human MCH1 receptor.
 151. The process ofany of claims 147, 148 or 150, wherein the cell is an insect cell. 152.The process of any of claims 147, 148 or 150, wherein the cell is amammalian cell.
 153. The process of any of claims 147, 146 or 150,wherein the cell is a mammalian cell which is nonneuronal in origin.154. The process of any of claims 147, 148 or 150, wherein the cell is aCOS-7 cell, a CHO cell, a 293 human embryonic kidney cell, a NIH-3T3cell, a mouse Y1 cell, or a LM(tk-) cell.
 155. A process for making acomposition of matter which specifically binds to a human MCH1 receptorwhich comprises identifying a chemical compound which specifically bindsto the human MCH1 receptor and then synthesizing the chemical compoundor a structural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting cellsexpressing on their cell surface the human MCH1 receptor, with both thechemical compound and a second chemical compound known to bind to thereceptor, and separately with only the second chemical compound, underconditions suitable for binding of both compounds, and detecting theextent of specific binding of the chemical compound to the human MCH1receptor, a decrease in the binding of the second chemical compound tothe human MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the human MCH1 receptor,wherein the cells do not normally express the human MCH1 receptor, thehuman MCH1 receptor is encoded by nucleic acid comprising the sequenceshown in FIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231(ATCC Accession No. 203157), and the second chemical compound is MCH ora homolog or analog of MCH.
 156. A process for making a composition ofmatter which specifically binds to a human MCH1 receptor which comprisesidentifying a chemical compound which specifically binds to the humanMCH1 receptor and then synthesizing the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as binding to the human MCH1 receptor bya process involving competitive binding which comprises contacting amembrane preparation from cells expressing on their cell surface thehuman MCH1 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and separately withonly the second chemical compound, under conditions suitable for bindingof both compounds, and detecting the extent of specific binding of thechemical compound to the human MCH1 receptor, a decrease in the bindingof the second chemical compound to the human MCH1 receptor in thepresence of the chemical compound indicating that the chemical compoundbinds to the human MCH1 receptor, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and thesecond chemical compound is MCH or a homolog or analog of MCH.
 157. Aprocess for making a composition of matter which is a human MCH1receptor antagonist which comprises identifying a chemical compoundwhich is a human MCH1 receptor antagonist and then synthesizing thechemical compound or a structural and functional analog or homologthereof, wherein the chemical compound is identified as a human MCH1receptor antagonist by a process which comprises contacting cellstransfected with and expressing DNA encoding the human MCH1 receptorwith the compound in the presence of a known human MCH1 receptoragonist, under conditions permitting the activation of the human MCH1receptor, and detecting a decrease in human MCH1 receptor activity, soas to thereby determine whether the compound is a human MCH1 receptorantagonist, wherein the cells do not normally express the human MCH1receptor, the human MCH1 receptor is encoded by nucleic acid comprisingthe sequence shown in FIG. 1 (Seq. ID No. 1) or contained in plasmidpEXJ.HR-TL231 (ATCC Accession No. 203197), and the known human MCH1receptor agonist is MCH or a homolog or analog of MCH.
 158. A processfor making a composition of matter which specifically binds to andinhibits the activation of a human MCH1 receptor which comprisesidentifying a chemical compound which specifically binds to and inhibitsthe activation of the human MCH1 receptor and then synthesizing thechemical compound or a structural and functional analog or homologthereof, wherein the chemical compound is identified as binding to andinhibiting the activation of the human MCH1 receptor by a process whichcomprises separately contacting cells expressing on their cell surfacethe human MCH1 receptor and producing a second messenger response uponactivation of the human MCH1 receptor, wherein such cells do notnormally express the human MCH1 receptor and the human MCH1 receptor isencoded by nucleic acid comprising the sequence shown in FIG. 1 (Seq. IDNo. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No.203197), with both the chemical compound and a second chemical compoundknown to activate the human MCH1 receptor, and with only the secondchemical compound, under conditions suitable for activation of the humanMCH1 receptor, and measuring the second messenger response in thepresence of only the second chemical compound and in the presence ofboth the second chemical compound and the chemical compound, a smallerchange in the second messenger response in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound indicating that the chemicalcompound inhibits activation of the human MCH1 receptor, wherein thesecond chemical compound is MCH or a homolog or analog of MCH.
 159. Theprocess of claim 158, wherein the second messenger response compriseschloride channel activation and the change in second messenger responseis a smaller increase in the level of inward chloride current in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound.
 160. Aprocess for preparing a composition which comprises identifying achemical compound which specifically binds to a human MCH1 receptor, andthen admixing a carrier and the chemical compound or a structural andfunctional analog or homolog thereof, wherein the chemical compound isidentified as binding to the human MCH1 receptor by a process involvingcompetitive binding which comprises contacting cells expressing on theircell surface the human MCH1 receptor, with both the chemical compoundand a second chemical compound known to bind to the receptor, andseparately with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting the extent ofspecific binding of the chemical compound to the human MCH1 receptor, adecrease in the binding of the second chemical compound to the humanMCH1 receptor in the presence of the chemical compound indicating thatthe chemical compound binds to the human MCH1 receptor, wherein thecells do not normally express the human MCH1 receptor, the human MCH1receptor is encoded by nucleic acid comprising the sequence shown inFIG. 1 (Seq. ID No. 1) or contained in plasmid pEXJ.HR-TL231 (ATCCAccession No. 203197), and the second chemical compound is MCH or ahomolog or analog of MCH.
 161. A process for preparing a compositionwhich comprises identifying a chemical compound which specifically bindsto a human MCH1 receptor, and then admixing a carrier and the chemicalcompound or a structural and functional analog or homolog thereof,wherein the chemical compound is identified as binding to the human MCH1receptor by a process involving competitive binding which comprisescontacting a membrane preparation from cells expressing on their cellsurface the human MCH1 receptor, with both the chemical compound and asecond chemical compound known to bind to the receptor, and separatelywith only the second chemical compound, under conditions suitable forbinding of both compounds, and detecting the extent of specific bindingof the chemical compound to the human MCH1 receptor, a decrease in thebinding of the second chemical compound to the human MCH1 receptor inthe presence of the chemical compound indicating that the chemicalcompound binds to the human MCH1 receptor, wherein the cells do notnormally express the human MCH1 receptor, the human MCH1 receptor isencoded by nucleic acid comprising the sequence shown in FIG. 1 (Seq. IDNo. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No.203197), and the second chemical compound is MCH or a homolog or analogof MCH.
 162. A process for preparing a composition which comprisesidentifying a chemical compound which is a human MCH1 receptorantagonist, and then admixing a carrier and the chemical compound or astructural and functional analog or homolog thereof, wherein thechemical compound is identified as a human MCH1 receptor antagonist by aprocess which comprises contacting cells transfected with and expressingDNA encoding the human MCH1 receptor with the compound in the presenceof a known human MCH1 receptor agonist, under conditions permitting theactivation of the human MCH1 receptor, and detecting a decrease in humanMCH1 receptor activity, so as to thereby determine whether the compoundis a human MCH1 receptor antagonist, wherein the cells do not normallyexpress the human MCH1 receptor, the human MCH1 receptor is encoded bynucleic acid comprising the sequence shown in FIG. 1 (Seq. ID No. 1) orcontained in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197), and theknown human MCH1 receptor agonist is MCH or a homolog or analog of MCH.163. A process for preparing a composition which comprises identifying achemical compound which specifically binds to and inhibits theactivation of a human MCH1 receptor, and then admixing a carrier and thechemical compound or a structural and functional analog or homologthereof, wherein the chemical compound is identified as binding to andinhibiting activation of the human MCH1 receptor by a process whichcomprises separately contacting cells expressing on their cell surfacethe human MCH1 receptor and producing a second messenger response uponactivation of the human MCH1 receptor, wherein such cells do notnormally express the human MCH1 receptor and the human MCH1 receptor isencoded by nucleic acid comprising the sequence shown in FIG. 1 (Seq. IDNo. 1) or contained in plasmid pEXJ.HR-TL231 (ATCC Accession No.203197), with both the chemical compound and a second chemical compoundknown to activate the human MCH1 receptor, and with only the secondchemical compound, under conditions suitable for activation of the humanMCH1 receptor, and measuring the second messenger response in thepresence of only the second chemical compound and in the presence ofboth the second chemical compound and the chemical compound, a smallerchange in the second messenger response in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound indicating that the chemicalcompound inhibits activation of the human MCH1 receptor, wherein thesecond chemical compound is MCH or a homolog or analog of MCH.
 164. Theprocess of claim 163, wherein the second messenger response compriseschloride channel activation and the change in second messenger responseis a smaller increase in the level of inward chloride current in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound.
 165. Theprocess of any of claims 155, 156, 157, 158, 160, 161, 162, or 163,wherein the cells is an insect cell.
 166. The process of any of claims155, 156, 157, 158, 160, 161, 162, or 163, wherein the cell is amammalian cell.
 167. The process of claim 166, wherein the mammaliancell is nonneuronal in origin.
 168. The process of claim 167, whereinthe nonneuronal cell is a COS-7 cell, a 293 human embryonic kidney cell,a CHO cell, a NIH-3T3. cell, a mouse Y1 cell, or a LM(tk-) cell.